Tissue-Specific Cross-Reactivity of Connexin32 Antibodies: Problems and Solutions Unique to the Central Nervous System

Figures & data

Figure 1. Schematic of the transmembrane structure of Cx32, highlighting the position of the peptides used to generate each of the antibodies assessed. Additional details are provided in Table 1.

Table 1 Connexin antibodies

Code	Catalogue number	Source	Epitope location^a	Type^b	[Western]	[Immuno]
AB1	CX32C13-M	Alpha Diagnostics	IL 110-128	Monoclonal	0.5 μg/ml	1:200
AB2	Hybridoma	David Paul	IL 95-125	Monoclonal	No dilution	No dilution
AB3	MAB3069	Chemicon	IL 95-125	Monoclonal	1:1000	1:100
AB4	13-8200	Zymed	IL^c	Monoclonal	1:250	1.0 μg/ml
AB5	71-0600	Zymed	IL^c	Polyclonal	1:125	2.0 μg/ml
AB6	C3470	Sigma	C-term 265-279	Polyclonal	1:200	1:600
AB7	C7854-05E	USBiological	C-term 19 aa	Polyclonal	0.7 μg/ml	1:400
AB8	C7854-04	USBiological	C-term	Polyclonal	3.0 μg/ml	1:800
AB9	34-5700	Zymed	C-term^d	Polyclonal	1:125	1.0 μg/ml
AB10	35-8900	Zymed	C-term^d	Monoclonal	3.0 μg/ml	1:800
AB11	51-2800 (Cx26)	Zymed	C-term	Polyclonal	1:125	NA
AB12	71-2200 (Cx30)	Zymed	C-term	Polyclonal	1:200	NA

NA, not applicable.

^aPeptides used to generate each antibody are listed by amino acid position where known (see Figure 1). Position and/or length of the peptides used to generate AB4, AB5, AB7-10 were not disclosed by the manufacturer. IL, intracellular loop; C-term, C-terminus.

^bAll monoclonal antibodies were raised in mouse. All polyclonal antibodies were raised in rabbit.

^cAB4 and AB5 were raised against the same proprietary IL antigen.

^dAB9 and AB10 were raised against the same proprietary C-term antigen.

Table 2 Antigenic sites along the IL and C-terminus of the Cx32 protein

	Site (amino acid position)^a	Sequence	Hopp-Woods	Kyte-Doolittle	Kolaskar and Tongaonkar
IL epitopes	118-125	EEVKRHKV	✓	✓
	98-105	QQHIEKKM	✓	✓
	109-116 (112-119)	EGHGDPLH	✓	✓	✓ (GDPLHLEE)
C-term epitopes	220-227	AQRRSNPP	✓	✓
	225-232	NPPSRKGS	✓	✓
	239-246 (235-242)	SPEYKQNE	✓	✓	✓ (FGHRLSPE)
	252-259	SEQDGSLK	✓	✓
	271-278	GLAEKSDR	✓	✓

Abbreviations are as in Table 1.

^aSites are listed from most to least likely to raise an antibody response determined using three different bioinformatic tools: (1) Antigenic Peptide Tool (Immunomedicine group at Universidad Complutense de Madrid; Kolaskar and Tongaonkar method); (2) Abie Pro 3.0 (Chang Biosciences; Hopp-Woods and Kyte-Doolittle hydrophilicities).

Figure 2. Four of the five Cx32 antibodies directed against the intracellular loop cross-react with a protein exhibiting the same mobility as Cx32 in Cx32^Y/− brain but not Cx32^Y/− liver under reducing/denaturing conditions. (A–E, left panel) Transmembrane schematics of the peptides for each Cx32 antibody, with the colored circles representing available information about each immunizing peptide sequence. Black brackets indicate primary sequence with the highest antigenic potential (see Table 2). (A–E, right panel) Cx32 immunoblots were performed using 10 μg liver or 30 μg of human (Hu) and murine hippocampus or brain lysates. Lines indicate size markers. Black arrowheads point to Cx32 specifically detected by all antibodies in liver (A–E) and AB5 in brain (E). White arrowheads indicate isoform variations specific to human samples (D, E). Arrows indicate the cross-reactive protein(s) detected by AB1 to AB4 in both Cx32^Y/+ brain tissue and the Cx32^Y/− control (A–D). Note that this particular cross-reactive protein(s) migrates with the same mobility as Cx32 when separated on 12.5% Tris-HCl polyacrylamide gels.

Figure 3. Four of the five Cx32 antibodies directed against the C-terminal tail cross-react with a protein exhibiting the same mobility as Cx32 in Cx32^Y/− brain but not Cx32^Y/− liver under reducing/denaturing conditions. As in Figure 2, lines indicate size markers. Closed arrowheads point to Cx32 specifically detected by all antibodies in liver (A–E) and AB10 in brain (E). White arrowheads indicate isoform variations specific to human samples (C–E). Arrows indicate the cross-reactive protein(s) detected by AB6-9 in both Cx32^Y/+ brain tissue and the Cx32^Y/− control (A–D). Note that this particular cross-reactive protein(s) migrates with the same mobility as Cx32 when separated on 12.5% Tris-HCl polyacrylamide gels. All other details are as in Figure 2.

Figure 4. Specific immunofluorescent detection of Cx32 in both liver and brain using intracellular loop-directed antibodies. (A–E) All five antibodies directed against epitopes localizing to the Cx32 intracellular loop detected Cx32 at hepatocyte plasma membrane in Cx32^Y/+ liver tissue (arrows, inset, Liver), with minimal to no background reactivity in the Cx32^Y/− controls with the exception of some low level background standing of hepatocyte plasma membrane with AB5 (arrowhead, Liver). (A, B, D, E) Four antibodies detected fixed protein in situ in Cx32^Y/+ mouse hippocampal sections with (D) AB4 providing the most robust signal. Hippocampal immunostaining was evident at the plasma membrane of cells with expected oligodendrocyte and/or oligodendrocyte precursor cell morphology (arrows, inset, Hippocampus). None of the antibodies exhibited significant cross-reactivity with Cx32^Y/− brain tissue. GrDG, granule cell layer of the dentate gyrus; PMNL, polymorphonuclear layer of the dentate gyrus; CA3c, CA pyramidal cell field 3c of the hippocampus. Scale bars, 50 μm.

Figure 5. Specific immunofluorescent detection of Cx32 in liver but not brain using C-terminal tail-directed antibodies. (A–E) All five C-terminal antibodies detected Cx32 at hepatocyte plasma membrane in Cx32^Y/+ liver tissue (arrows, inset, Liver), with minimal to no background reactivity in the Cx32^Y/− controls. No specific immunosignal was seen in the Cx32^Y/+ hippocampus under the specific fixation and processing conditions employed in this study. Abbreviations are as in Figure 4. Scale bars, 50 μm.

Figure 6. Brain-specific cross-reactivity is not observed following immunoprecipitation. (A) Cx32 lysates were purified from native RIPA lysates using monoclonal AB1, resolved under denaturing conditions by SDS-PAGE, and immunoblotted using polyclonal AB6. In the first two lanes WT (Cx32^Y/×) and null-mutant (Cx32^Y/−) liver lysates were subjected to Western analysis under reducing/denaturing conditions. The following three lanes represent human hippocampus and murine brain samples from WT (Cx32^Y/+) and null-mutant (Cx32^Y/−) controls immunoprecipitated with AB1 under native conditions before denaturing/reducing SDS-PAGE separation and immunoblotting with AB6. Specific immunoaffinity purification of Cx32 is observed under these conditions. (B) Standard Western analysis with aliquots of the same samples used in (A) reiterate the cross-reactivity observed when protein is first detected under reducing/denaturing conditions.

Figure 7. Sucrose gradient fractionation reveals that the brain-specific cross-reactive protein is approximately 4 kDa smaller than Cx32 and exhibits a distinct subcellular localization. (A–D) Total tissue lysates (T) or the Triton X-100–insoluble pellet (P) and (E) fractions 1 to 10 obtained through sucrose fractionation were immunoblotted under denaturing/reducing conditions using AB1. To achieve maximal separation, proteins were separated on 15% Tris-HCl polyacrylamide gels. Each lane contains 5 μg of protein. (A) Sucrose gradient fractions of Cx32^Y/+ liver are compared to total tissue lysates of Cx32^Y/+ liver or brain. Note the ∽4-kDa size difference between the species predominating in Cx32^Y/+ liver compared to brain. (B) Sucrose gradient fractions of Cx32^Y/− liver are compared to total tissue lysates of Cx32^Y/+ liver or brain. No signal was detected in Cx32^Y/− control lysates. (C) Sucrose gradient fractions of Cx32^Y/+ brain are compared to total brain lysates prepared from Cx32^Y/+ and Cx32^Y/− mice (top panel). Exposure times were extended from that shown in A and B to enable detection of endogenous Cx32 in fractions 4 and 5 (arrow), migrating 4 kDa higher than the cross-reactive protein that was present at higher abundance and enriched in fractions 6 to 10. Lower panels characterize each fraction using organelle-specific markers: mitochondria (coxIV), lipid rafts (caveolin-1 and flotillin-1), plasma membrane (syntaxin-1), lysosomes (LAMP-1), and the trans-Golgi network (Golgin-97). The fractionation of liver tissue exhibited the same pattern of organelle-specific immunoreactivity (data not shown). (D) Only the lower molecular weight cross-reactive band was present in sucrose gradient fractions prepared from Cx32^Y/− mouse brain. (E) Schematic of the sucrose fractionation showing the gradients enriched for Cx32 in both brain and liver.

Figure 8. Cx32 antibodies do not cross-react with Cx26, Cx29, or Cx30 under denaturing/reducing conditions. (A) Liver and brain samples were prepared from wild-type (Cx32^Y/×), Cx32^Y/−, and Cx29^−/−. Cx32^Y/−/Cx29^−/−, or Cx30^−/− null-mutant mice, separated on 15% Tris-HCl polyacrylamide gels, and immunoblotted for Cx32 using monoclonal AB1. To distinguish between the cross-reactive protein(s) and endogenous Cx32 in brain samples, fraction 5 lysate from sucrose gradient separations (see Figure 7) of WT (Cx32^Y/+) was included as a positive control. All brain samples, except WT brain fraction 5, exhibited the lower molecular weight cross-reactive band. Arrowheads indicate Cx32; arrows indicate the cross-reactive (CR) protein. (B) Immunoblotting of Cx30 in lysates prepared from human brain, wild-type mouse brain and liver, and Cx30^−/− brain confirmed the presence of Cx30 in CNS tissue (arrowhead), absent from liver. Specificity was established using null-mutant controls. (C) Cx26 protein, present in Cx32^Y/+ brain and liver (arrowhead), was below detection levels in Cx32^Y/− tissue.