Identification of kinases, phosphatases, and phosphorylation sites in human and porcine spermatozoa

Abstract

Multiple inter-connected signaling pathways, involving kinases and phosphatases, form a framework that controls sperm motility, function, and fertilizing ability. Methods that give a broad view of the proteomic landscape may prove valuable in uncovering new crosstalk connections, as well as in discovering new proteins within this regulatory framework. A multi-immunoblotting strategy was utilized to evaluate this concept on human and porcine spermatozoa samples. In human and porcine spermatozoa, a diversity of kinases were identified including protein kinase A (PKA), protein kinase B (PKB), isoforms of protein kinase C (PKC), calmodulin-dependent kinases (CAMK), casein kinase (CK), and isoforms of glycogen synthase kinase (GSK3). Several phosphatases, such as protein phosphatase (PP)-1, PP2A, PP2C, and mitogen activated protein kinase (MAPK) phosphatase (MKP-1), were identified in human spermatozoa. The phosphorylation epitopes recognized belonged to members of the MAPK family, in addition to α and β isoforms of GSK3 and cAMP response element binding protein (CREB). Proteomic approaches that allow a broad view may aid in understanding the crosstalk between signaling systems in spermatozoal physiology.

• Kinase
• phosphatase
• phosphorylation
• spermatozoa

Introduction

Signal transduction pathways involving multiple kinases and phosphatases form a framework that controls sperm motility, function, and fertilizing ability. The effects of calcium and cAMP-dependent proteins on spermatozoal activity are well established [Brokaw 1987; De Jonge 1996; Doherty et al. 1995; Talbot et al. 1976]. Protein kinase B (PKB/Akt) has been more recently recognized as another important component in the signaling cascades that direct sperm physiology [Aquila et al. 2004; Gallardo Bolanos et al. 2014; Jungnickel et al. 2007; Nauc et al. 2004; Vadnais et al. 2013]. Evidence that Akt may affect sperm behavior stemmed from work with wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor [Fisher et al. 1998]. This protein, PI3K, was known to act as an upstream regulator of Akt. Additional studies uncovered further clues for Akt involvement by identifying a downstream target, glycogen synthase kinase 3 (GSK3), present in spermatozoa [Vijayaraghavan et al. 1996; Vijayaraghavan et al. 2000]. The acquisition of sperm motility was associated with decreased GSK3 activity [Vijayaraghavan et al. 1996]. Thus, two components in the PI3K-Akt-GSK3 pathway were known to be present in sperm, with Akt only later being identified [Aquila et al. 2004; Nauc et al. 2004]. In addition to protein kinases, the phosphatases that modulate these kinases have a vital role in sperm physiology [Rotfeld et al. 2014; Signorelli et al. 2012]. A relationship was noted between protein phosphatase-1 (PP-1) and GSK3 [Vijayaraghavan et al. 1996]. Future studies would reveal further connections and help to solve this physiological puzzle, with PP-1 acting as a negative regulator by dephosphorylating Akt, a negative regulator of GSK3 [Thayyullathil et al. 2011]. Mathematically, one would expect an increase to result from the removal of a negative (subtracting a negative). Experiments on sperm from mice lacking Akt corroborated these deductions of elevated GSK3 with impaired motility and fertility [Kim et al. 2012]. Building on the work of these studies, the importance of this pathway and its ability to crosstalk with other signaling cascades are better understood [Aparicio et al. 2007; Bragado et al. 2010; Somanath et al. 2004].

Methods that give a broad view of the proteomic landscape may prove valuable in filling in these missing pieces by uncovering new connections as well as discovering new proteins. Previous research in our laboratory considering similarities in growth factors and signaling proteins between humans and farm animals lead to the investigation of boar and human spermatozoa in a multi-immunoblotting strategy offered by Kinexus [Henricks et al. 1998]. Both boar and human spermatozoa were analyzed for kinases and phosphorylated proteins. Human sperm samples were also processed for phosphatase identification. This strategy can also be adapted to aid in elucidating the activities of compounds and proteins that affect fertility and reproduction in a variety of species.

Results and Discussion

Kinases in porcine spermatozoa

Immunoblot scans of the 28 proteins identified by the Kinetworks™ Protein Kinase Screen (KPKS) in porcine are displayed in Figure 1. Table 1 lists the location of the kinases shown in Figure 1, as well as their molecular weight and relative lane abundance. Protein kinase B (PKB/Akt) was identified in this screen as well as its downstream targets GSK3α and GSK3β. Other proteins activated by phosphoinositides include PKCλ/ι and PKCζ. Calcium and cAMP regulated proteins including calmodulin-dependent kinases (CAMK) and protein kinase A (PKA) were identified, respectively. Additionally, multiple members of the casein kinase 2 (CK2) and ribosomal S6-kinase (RSK) families were present.

Figure 1. Kinetworks™ Protein Kinase Screen (KPKS) of porcine spermatozoa. Known proteins are marked with an arrow and labeled with a ‘p’ for porcine and a corresponding number which can be used with the key for identification. A: proteins p1-p18 and B: proteins p19-p28.

Table 1. Kinases in porcine spermatozoa.

ID	Lane	Band	Protein	Key	MW	Qty
p1	2	1	v-raf sarcoma viral oncogene B1	RAFB	99	1.5
p2	2	2	Extracellular regulated kinase 3 (56)	ERK3	55	9.6
p2	2	3	Extracellular regulated kinase 3 (52)	ERK3	52	9.3
p3	3	1	Protein kinase B alpha	PKBa	60	3.9
p4	6	6	p38 Hog MAP kinase	p38	38	4.1
p5	6	7	Cyclin-dependent kinase 5	CDK5	23	5.0
p6	11	3	Protein kinase C lambda/iota	PKCl	60	5.7
p7	11	6	Stress activated protein kinase (JNK) (45)	SAPK	48	4.2
p7	11	8	Stress activated protein kinase (JNK) (38)	SAPK	40	1.2
p8	12	3	Extracellular-regulated MAP kinase 6	ERK6	47	6.4
p9	13	1	Protein kinase C zeta (88)	PKCz	87	7.0
p9	13	2	Protein kinase C zeta (78)	PKCz	80	10.2
p10	13	3	MAP Kinase Kinase 1 (MKK1)	MEK1	42	3.1
p11	14	2	p21 activated kinase 1	PAK1	64	4.8
p12	15	2	MAP kinase kinase 4	MKK4	38	2.5
p13	16	9	MAP Kinase Kinase 6	MKK6	31	0.8
p14	18	1	Glycogen synthase kinase 3 alpha	GSK3a	45	47.0
p15	18	2	Glycogen synthase kinase 3 beta	GSK3b	41	17.3
p16	19	2	Ribosomal S6 kinase 1	RSK1	75	9.0
p17	20	2	Ribosomal S6 kinase 2	RSK2	74	2.5
p18	20	6	Casein kinase 2 (39)	CK2	41	24.8
p18	20	7	Casein kinase 2 (37)	CK2	38	9.7
p18	20	8	Casein kinase 2 (35)	CK2	34	2.5
p19	3	9	Lymphocyte-specific kinase	LCK	44	4.2
p20	5	1	Death associated protein kinase 1	DAPK	160	1.0
p21	5	5	Oncogene Lyn	LYN	48	6.6
p22	10	3	Calmodulin-dependent kinase 4	CaMK4	62	5.7
p23	11	2	Calmodulin-dependent kinase 1	CaMK1	44	7.4
p24	12	2	Calmodulin-dependent kinase kinase	CaMKK	48	3.8
p25	13	1	NIMA-related kinase 2	NEK2	47	8.4
p26	15	4	Protein kinase A	PKA	37	15.7
p27	16	1	Focal adhesion kinase	FAK	127	5.0
p28	18	4	ZIP kinase	ZIP	45	16.8

ID: location of proteins on Figure 1. Lane: each gel is divided into lane numbers 1 to 21. Lanes 1 and 21 are used for the molecular weight standards. Band: identifies the location of each detected protein in each lane. Protein: common protein name, isoforms are denoted by (#). Key: common protein name abbreviation. MW: the molecular mass is the protein size in KDa as measured by its location on the gel. QTY: relative quantity for a protein in its respective lane.

Proteins in human spermatozoa

Immunoblot scans of the 34 proteins identified by the KPKS screen in human sperm are displayed in Figure 2. Table 2 lists the location of the kinases in Figure 2, as well as their molecular weight and relative lane abundance. As in porcine sperm, Akt and GSK3α and β were identified. In addition the upstream Akt regulator phosphoinositide-dependent kinase (PDK1) was identified. Multiple members of PKC, ERK, MAPK, CAMK, and CK2 families were identified in human sperm. Figure 2C, is a blot of Akt identified in human sperm by two different antibodies.

Figure 2. Kinetworks™ Protein Kinase Screen (KPKS) of human spermatozoa. Known proteins are marked with an arrow and labeled with an ‘h’ for human and a corresponding number which can be used with the key for identification. A: proteins h1-23 and B: proteins h24-34. Results of Western blot: Lanes 1 and 2 were incubated with Ab1 and Lanes 3 and 4 were incubated with Ab2. Lanes 1 and 3 are extracts of HCT-8 cells used as positive controls. Lanes 2 and 4 are sperm extracts.

Table 2. Kinases in human spermatozoa.

ID	Lane	Band	Protein Name	KEY	MW	Qty
h1	2	1	v-raf murine sarcoma viral oncogene B1	RAFB	89	6.5
h2	2	2	Extracellular regulated kinase 3	ERK3	54	16.6
h3	3	1	Protein kinase B alpha	PKBa	59	5.1
h4	3	2	Extracellular regulated kinase 1	ERK1	40	15.4
h5	3	3	Extracellular regulated kinase 2	ERK2	38	19.9
h6	4	2	3-phosphoinositide dependent kinase 1	PDK1	52	1.2
h7	4	4	Extracellular regulated kinase 2	ERK2	38	7.8
h8	5	1	Protein kinase C mu	PKCm	117	2.3
h9	6	5	p38 Hog MAP kinase	p38	38	8.9
h10	6	7	Cyclin-dependent kinase 5	CDK5	23	3.3
h11	11	1	Protein kinase C lambda/iota	PKCl	61	6.5
h12	11	3	Stress activated protein kinase (JNK) (45)	SAPK	48	7.2
h12	11	4	Stress activated protein kinase (JNK) (38)	SAPK	40	1.0
h13	12	2	Protein kinase C theta	PKCt	77	6.5
h14	12	5	Extracellular-regulated MAP kinase 6	ERK6	46	2.8
h15	13	1	Protein kinase C zeta	PKCz	78	37.0
h16	13	5	MAP kinase kinase 1	MEK1	41	5.7
h17	14	6	MAP kinase kinase 2	MEK2	41	3.6
h18	15	1	MAP kinase kinase 4	MKK4	38	2.6
h19	16	1	MAP kinase kinase 6	MKK6	31	6.3
h20	18	1	Glycogen synthase kinase 3 alpha	GSK3a	45	15.7
h21	18	2	Glycogen synthase kinase 3 beta	GSK3b	40	7.6
h22	19	1	Ribosomal S6 kinase 1	RSK1	76	1.4
h23	20	3	Casein kinase 2 (39)	CK2	39	20.6
h23	20	4	Casein kinase 2 (37)	CK2	38	15.9
h23	20	5	Casein kinase 2 (35)	CK2	34	21.1
h24	5	1	Death associated protein kinase 1	DAPK	154	5.1
h25	5	4	Oncogene Lyn	LYN	48	1.5
h26	6	1	Spleen tyrosine kinase	SYK	68	4.2
h27	11	2	Calmodulin-dependent kinase 1	CaMK1	43	3.1
h28	12	1	Calmodulin-dependent kinase kinase	CaMKK	51	5.4
h29	13	1	NIMA-related kinase 2	NEK2	50	5.1
h30	14	2	Oncogene Raf1 (70)	RAF1	73	5.1
h30	14	3	Oncogene Raf 1 (61)	RAF1	66	3.3
h31	15	2	Protein kinase A	PKA	36	11.6
h32	16	1	Focal adhesion kinase	FAK	120	3.3
h33	17	1	G protein-coupled receptor kinase 2	GRK2	72	6.5
h34	18	2	ZIP kinase (48)	ZIP	44	3.3
h34	18	3	ZIP kinase (45)	ZIP	41	3.3

ID: location of proteins on Figure 2. Lane: each gel is divided into lane numbers 1 to 21. Lanes 1 and 21 are used for the molecular weight standards. Band: identifies the location of each detected protein in each lane. Protein: common protein name with isoforms denoted by (#). Key: common protein name abbreviation. MW: the molecular mass is the protein size in KDa as measured by its location on the gel. QTY: relative quantity for a protein in its respective lane.

Phosphatases and phosphorylation sites

Table 3 lists the identities of the phosphatases and their location shown in Figure 3A. Multiple phosphatases from human sperm were identified including protein phosphatase (PP-1), PP2A, PP2C, and MAP kinase phosphatase (MKP-1). Phosphorylation epitopes recognized in Figure 3B are listed in Table 3 and include several members of the MAPK family, in addition to α and β isoforms of GSK3 and the multi-functional cAMP response element binding protein (CREB).

Figure 3. Kinetworks™ Protein Phosphatase Screen (KPPS) phosphatase and Kinetworks™ Phosphosite Screen (KPSS) phosphorylation site screens of human spermatozoa. Known proteins are marked with an arrow and labeled with a ‘s’ for phosphatase or a ‘k’ for phosphorylation and a corresponding number which can be used with the Key for identification. A: phosphatases along with B: phosphorylated proteins are indicated.

Table 3. Results from phosphatase and phosphorylation site screens.

ID	Lane	Band	Protein Name	KEY	Type	MW
s1	2	1	Protein phosphatase 2A – A	PP2A/A	PSTP	48
s2	4	1	Protein phosphatase 2C – d isoform	PP2C/d	PSTP	45
s3	6	1	Protein phosphatase 2C – b isoform	PP2C/b	PSTP	47
s4	8	1	MAP kinase phosphatase 1	MKP-1	PTYP	39
s5	10	2	Protein phosphatase 1	PP1/C-a	PSTP	30
k1	3	4	Cyclin-dependent kinase 1	CDK1	Y15	26
k2	4	3	p38 alpha MAP kinase	p38	T180/Y182	38
k3	8	2	Extracellular regulated kinase 2	ERK2	T185/Y187	38
k4	17	7	Glycogen synthase kinase 3 alpha	GSK3a	Y279	44
k5	17	8	Glycogen synthase kinase 3 beta	GSK3b	Y216	38
k6	19	2	MAP kinase kinase 1/2	MEK1/2	S221/S225	39
k7	20	5	cAMP Response Element Binding Protein	CREB	S133	43

ID: location of phosphatases (s) or phosphorylation site (k) on Figure 3. Lane: each gel is divided into lane numbers 1 to 21. To conserve figure space, empty lanes were removed from the figure (Lanes 11–21 on A and Lanes 6–8 and 10–15 on B). Band: identifies the location of each detected protein in the lane. Key: PSTP: protein-serine/threonine phosphatase; PTYP: protein-threonine/tyrosine phosphatase. For the phosphorylation site screen the key refers to the epitope(s) of the target protein. MW: the molecular mass is the protein size in KDa as measured by its location on the gel. QTY: relative quantity for a protein in its respective lane.

Protein expression patterns in human and porcine sperm

Table 4 presents a qualitative comparison of protein expression in porcine and human sperm (Supplemental Table 1 cross-references mouse proteins). Proteins with the greatest band intensity were normalized to 100 and other proteins were expressed as a percentage to establish relative abundance. Because of possible differences in antibody and species sensitivities strict quantitative analyses is not advisable without controlling for these variables. A recent review explores the role of these proteins in kinase cascades in several species [Lackey and Gray 2015].

Table 4. Comparison of protein expression patterns in human and porcine sperm.

Protein	ID	Human	Porcine
3-phosphoinositide dependent protein kinase	PDK1	1	1
Protein kinase B alpha	PKBa	1	2
Glycogen synthase kinase 3 alpha	GSK3a	21	79
Glycogen synthase kinase 3 beta	GSK3b	10	29
Calmodulin-dependent kinase 1	CaMK1	3	1
Calmodulin-dependent kinase 4	CaMK4		2
Calmodulin-dependent kinase kinase	CaMKK	4	1
Casein kinase 2	CK2 (35)	100	10
Casein kinase 2	CK2 (37)	75	39
Casein kinase 2	CK2 (39)	98	100
Cyclin-dependent kinase 5	CDK5	4	9
Extracellular regulated kinase 1	ERK1	4
Extracellular regulated kinase 2	ERK2	6
Extracellular regulated kinase 3	ERK3	18	16
Extracellular-regulated MAP kinase 6	ERK6	1	3
Focal adhesion kinase	FAK	5	2
G protein-coupled receptor kinase 2 (BARK2)	GRK2	8
MAP Kinase Kinase 1 (MKK1)	MEK1	4	1
MAP kinase kinase 2 (MKK2)	MEK2	3
MAP kinase kinase 4 (MKK4)	MKK4	6	8
MAP Kinase Kinase 6 (MKK6)	MKK6	2	2
NIMA (never in mitosis)-related kinase 2	NEK2	3	1
Oncogene Lyn	LYN	1	2
Oncogene Raf1	RAF1	12
p21 activated kinase 1	PAK1		3
p38 Hog MAP kinase	p38	11	7
Protein kinase A	PKA	22	6
Protein kinase C lambda/iota	PKCl	6	7
Protein kinase C mu	PKCm	2
Protein kinase C theta	PKCt	3
Protein kinase C zeta	PKCz	27	3
Ribosomal S6 kinase 1	RSK1	5	10
Ribosomal S6 kinase 2	RSK2		10
Stress activated protein kinase (JNK)	SAPK (45)	7	5
v-raf murine sarcoma viral oncogene homolog B1	RAFB	7	2
ZIP kinase (death associated protein kinase 3)	ZIP (45)	8	16

The trace quantity of a band in the Kinetworks™ Protein Kinase Screen (KPKS) as measured by the area under the intensity profile curve was normalized to 100. For human sperm, casein kinase CK2 (35) with a trace quantity of 37,474 and for porcine sperm, CK2 (39) with a trace quantity of 44,571 were normalized to 100. Because of the potential antibody differences and species sensitivity a quantitative comparison is not recommended.

The multi-immunoblotting strategy used in Kinexus screens offers time and cost savings when trying to visualize the proteomic landscape. In the screen for kinases in boar and human sperm samples, components of multiple signaling cascades were identified that are known to be regulators of sperm physiology. Proteins identified in the kinase screen included members of PKA, PKC, and PKB/Akt pathways and regulators such as CAMK, ERKs, and MAPKs [Almog et al. 2008; Bernabò et al. 2010; Bragado et al. 2010; Gallardo Bolanos et al. 2014; González-Fernández et al. 2013; Mannowetz et al. 2010]. Ribosomal S6 kinases were identified in porcine sperm and human sperm [Sagare-Patil and Modi 2013]. Cyclin-dependent kinase 5, CK2, and RAFB were also identified for the first time in porcine spermatozoa. In addition to the qualitative results, semi-quantitative data could be acquired in the future by calibration to different quantities of protein extract loaded per lane. Future research could ascertain if the CK2 abundance is associated with fertility. In addition, specific preparative methods could be used to enrich cellular components such as the mitochondrial fractions or membranes to complement the immunocytochemistry and better understand the activities of these proteins in affecting mitochondrial and sperm function [Gallardo Bolanos et al. 2014]. Comparisons of protein expression from total sperm with swim-up selected and low-motility populations could also be screened to better understand differences between high-fertility and lower-fertility samples. Other approaches can also give views of the proteomic landscape such as a phosphoproteomic UPLC-MS method that identified multiple proteins altered in asthenozoospermia [Parte et al. 2012]. Among these proteins with altered expression were PKA and Akt [Parte et al. 2012]. The phosphatase screen identified multiple phosphatases that regulate the activity of these protein kinases, including serine-threonine phosphatases, such as PP-1, PP2A, and PP2C as well as MKP-1, a dual specificity phosphatase (DUSP) that targets threonine and tyrosine residues. In the screen of phosphorylation sites, members of cAMP, MAPK, and AKT-GSK3 pathways were identified. The expression patterns for both GSK3 isoforms in human and porcine sperm appears to follow that of bovine sperm with the α isoform having higher expression [Somanath et al. 2004]. Screening of this type may yield interesting data when comparing samples of high and low fertility. Furthermore, several unidentified phosphorylated proteins were detected after progesterone and IGF-stimulation. Subsequent isolation of these unidentified proteins combined with mass-spectrometric sequencing could be utilized to identify new proteins and reveal new interactions. The multiple signaling pathways in human and porcine spermatozoa identified using the approach described may help in understanding the complex relationships between these systems in male reproductive physiology.

Materials and Methods

Spermatozoa

Discarded frozen samples provided by the ART Laboratory at Greenville Health System came from anonymized donors ranging in age from 25 to 40 y who were free of HIV and hepatitis C. Because no patient data were involved, this study did not require Internal Review Board approval. Samples used had no extender other than glycerol and were thawed in a water bath at 40 °C for 5 min. Three samples of good motility (>75%) were pooled and extracted. Sperm concentration and motion parameters were assessed with the IVOS semen analyzer (Hamilton Thorne Research, Beverly, MA, USA).

Boar spermatozoa were obtained from high-fertility Duroc breed animals through Lean Value Sires (New Carlisle, OH, USA). Semen was shipped overnight to the laboratory and packaged so the temperature was maintained at approximately 17 °C. Methods for preparing the spermatozoa utilized non-toxic plastics [Johnson et al. 1996; Mortimer 1994]. The semen from several boars was pooled and centrifuged at 400 × g for 5 min to remove the extender. Spermatozoa samples were washed twice with 0.2 mM NaHCO₃ Tyrode's Albumin-Lactate-Pyruvate Media (TALP) and resuspended in 0.5 mL media [Gray et al. 2015].

Processing

Motile human and boar spermatozoa were each selectively separated from the pellet using the swim-up method for immunoblotting analysis to enrich low-abundance proteins [Lackey et al. 1998; Mortimer 1994]. The supernatants containing the motile spermatozoa were carefully pipetted, pooled, and processed [Gray et al. 2015; Henkel and Schill 2003; Lalancette et al. 2006].

Progesterone and IGF-1 reagents were purchased from Sigma (St. Louis, MO, USA). Sperm were incubated for 30 min at 37 °C with 100 ng/mL IGF-1 and 1 µg/mL progesterone added to TALP for induction and analysis in the Kinetworks™ Phosphosite Screen (KPSS) phosphorylation site screen [Blackmore et al. 1990; Kaya et al. 2012; Oberlender et al. 2013]. Induced and non-induced swim-up sperm samples were prepared for Western blotting or Kinexus analysis after being lysed in 500 µL of Cytobuster reagent per mL of sperm pellet (EMD Millipore, Billerica, MA, USA) [Martins-Branco et al. 2012]. In addition, protease and phosphatase inhibitors were added to the lysis mixture. Phosphatase I and II inhibitors were from Sigma and the Halt protease inhibitor was from Thermo-Fisher (Rockford, IL, USA), both were used according to manufacturer’s instructions.

Protein identification

Secondary antibodies, transfer membranes, reagents, and film used for detection were purchased from Thermo-Fisher. Human lysates were quantified by the Bradford protein method and after boiling for 5 min, 30 μg of protein was separated under denaturing SDS-PAGE on 10% polyacrylamide Tris-glycine gels (Bio-Rad, Hercules, CA, USA), and then electroblotted to PVDF membranes [Aquila et al. 2004; Gallardo Bolanos et al. 2014; Vijayaraghavan et al. 2000]. Non-specific sites were blocked with 5% non-fat dry milk in 0.2% Tween-20 in Tris-buffered saline (TBST) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C. A primary mouse anti-Akt antibody (Ab1) against the pleckstrin homology domain corresponding to residues 1-149 of human Akt1 was purchased from EMD Millipore (Billerica, MA, USA) and used at a dilution of 1:500. A primary rabbit anti-Akt antibody against amino acids 345-480 of Akt1/2 of human origin (Ab2) was acquired from Santa Cruz Biotechnology (Dallas, TX, USA) and used at dilution of 1:200. Protein extracts from the HCT-8 cell line were used as a positive control. Membranes were incubated with their respective goat-anti-rabbit or goat-anti-mouse HRP-labeled secondary antibodies at room temperature for 1 hr. Secondary antibodies were detected with SuperSignal™ West Femto Maximum Sensitivity Substrate according to the manufacturer's instructions. Each membrane was exposed CL-XPosure films for 2 min [Rago et al. 2014].

Human and porcine sperm samples were analyzed in KPSS and KPKS; human samples were also subjected to KPPS (see http://www.kinexus.ca for more information). A total of 600 µg lysate was prepared for KPKS and KPPS and 350 µg for KPSS [Pelech 2004]. Briefly, a multi-immunoblot system is applied using screened antibodies for protein identification and detection [Pelech et al. 2003; Pelech 2004]. After chemiluminescent detection the protein signals are quantified by an optical scanner and results provided in image and table formats [Pelech et al. 2003; Pelech 2004].

Supplementary material available online

Acknowledgments

The authors wish to acknowledge Dr. William Boone for assistance and access to samples and Dr. Donald M. Henricks for guidance and expertise.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Author contribution

Conceived and designed the experiments: BL, SG; Performed the experiments: BL, SG; Analyzed the data: BL,SG; Contributed reagents and materials: SG; Both authors wrote the manuscript and approved the revised paper.