Toll-like Interleukin -1 Receptor Regulator (TILRR) Protein, a Major Modulator of Inflammation, is Expressed in Normal Human and Macaque Tissues and PBMCs

Figures & data

Table 1 Adult Human Normal Tissue Lysates and Monkey Normal Tissue Cell Lysates

Human Normal Tissue Lysates (Supplier: Novus Biologicals, Bio-Techne Canada)
	Tissue Lysates	Catalog #		Lot#	Sex	Age (Years)		Single Donor?
1	Human Trachea Whole Tissue Lysate (Adult Whole normal)	NB820- 59241		B204077	Female	82		Yes
2	Human Lung whole tissue lysate (Adult whole normal)	NB820-59239		C107099	Female	70		Yes
3	Human Stomach Whole Tissue Lysate (Adult Whole normal)	NB820-59260		C603538	Female	83		Yes
4	Human Small intestine (Ileum) Whole Tissue Lysate (Adult Whole normal)	NB820-59255		C411100	Male	62		Yes
5	Human Large Intestine Cecum Whole Tissue Lysate (Adult Whole normal)	NB820-59204		B204091	Male	26		Yes
6	Human Colon Whole Tissue Lysate (Adult Whole normal)	NB820-59205		C311229	Female	60		Yes
7	Human Rectum Whole Tissue Lysate (Adult Whole normal)	NB820-59250		B604180	Female	83		Yes
8	Human Vagina Whole Tissue Lysate (Adult Whole normal)	NB820-59278		A603644	Female	69		Yes
9	Human Cervix Whole Tissue Lysate (Adult Whole normal)	NB820-59275		C210005	Female	42		Yes
10	Human Uterus Whole Tissue Lysate (Adult Whole normal)	NB820-59274		A312043	Female	40		Yes
11	Human Penis Whole Tissue Lysate (Adult Whole normal)	NB820-59247		A207007	Male	n/a		Yes
Cynomolgus Monkey Normal Tissue Cell Lysates
	Tissue Cell Lysates		Monkey ID#		Sex		Age (Years)
1	Adult Cynomolgus monkey normal lungs		AM271		Female		14
2	Adult Cynomolgus monkey normal colon		AM271		Female		14
3	Adult Cynomolgus monkey normal rectum		AM271		Female		14
4	Adult Cynomolgus monkey normal lungs		BM843		Female		15
5	Adult Cynomolgus monkey normal colon		BM843		Female		15
6	Adult Cynomolgus monkey normal rectum		BM843		Female		15

Notes: All human tissue lysates were processed by using a total protein extraction kit (Catalog# NBP2-37853). Donors’ sex, age, and other information were obtained from the supplier (Novus Biologicals). ID#, identification number.

Table 2 Adult Human and Monkey Normal PBMCs Lysates

Human PBMCs Lysates
	Cell Lysates	ML#	Sex	Age (Years)
1	Human PBMCs	1260	Female	36
2	Human PBMCs	893	Female	53
3	Human PBMCs	1800	Female	28
4	Human PBMCs	2006	Female	40
Monkey PBMCs
	Cell Lysates	Monkey ID#	Sex	Age (Years)
1	Cynomolgus monkey PBMCs	AM271	Female	14
2	Cynomolgus monkey PBMCs	AT45	Female	13
3	Cynomolgus monkey PBMCs	BM796	Female	15
4	Cynomolgus monkey PBMCs	BM805	Female	11
5	Rhesus monkey PBMCs	R223	Female	6
6	Rhesus monkey PBMCs	R232	Male	6
7	Rhesus monkey PBMCs	R236	Male	6
8	Rhesus monkey PBMCs	R263	Male	6

Abbreviations: PBMCs, peripheral blood mononuclear cells; ML#, participant’s identification number; ID#, identification number.

Figure 1 TILRR protein expression in normal human tissues. Tissue lysates (2.5 µg/lane protein) were examined by Western blot (green color, left) and Coomassie blue staining (red color, right). TILRR protein was probed by primary mouse anti-TILRR F218G4 mAb followed by secondary goat anti-mouse IRDye 800CW antibody (1:10,000; LI-COR). TILRR protein bands (70 kDa, green color, cropped) are shown on the top of the bar graph (see Figure S1A for un-cropped blot image). Untransferred proteins in the gel after iBlot transfer were stained with Coomassie blue staining (G-250, Bio-Rad) and the protein bands (~70 kDa, red color, cropped) are shown on the top of the bar graph (see Figure S1B for un-cropped blot image). The bar graph shows the TILRR protein intensity (R.F.U) for each tissue lysate (n=1) where X-axis demonstrates the normal human tissue lysates and Y-axis indicates the signal intensity (R.F.U). kDa, kiloDalton; RFU, relative fluorescence units; S. intestine, small intestine (ileum).

Figure 2 TILRR protein expression in normal Cynomolgus monkey tissues. (A) Tissue lysates (5.0 µg/lane proteins) were examined by Western blot (green color, left) and Coomassie blue staining (red color, right). TILRR protein was probed by primary mouse anti-TILRR F218G4 mAb followed by secondary goat anti-mouse IRDye 800CW antibody (1:10,000; LI-COR). TILRR protein bands (70 kDa, green color, cropped) are shown on the top of the bar graph (Figure S1C shows the un-cropped blot image). Untransferred proteins in the gel after iBlot transfer were stained with Coomassie blue staining (G-250, Bio-Rad) and the protein bands (~70 kDa, red color, cropped) are shown on the top of the bar graph (Figure S1D shows the un-cropped gel). The bar graph indicates the TILRR protein intensity (R.F.U) for each tissue lysate (n=1). (B) TILRR protein intensity (R.F.U) (median with interquartile range [IQR]) of each tissue cell lysate observed in two cynomolgus monkeys (n=2). The X-axis represents the normal Cynomolgus monkey tissue cell lysates and Y-axis shows the signal intensity (R.F.U). AM271 and BM843 are the monkeys’ identification numbers. kDa, kiloDalton; RFU, relative fluorescence units; n= number of subjects.

Figure 3 TILRR protein expression in normal human and macaque PBMCs. (A) PBMC lysates (14.0 µg/lane proteins) of humans (n=4), rhesus monkeys (n=4), and cynomolgus monkeys (n=4) were analyzed by Western blot (green color, left) and Coomassie blue staining (red color, right). TILRR protein was probed by primary mouse anti-TILRR F218G4 mAb followed by secondary goat anti-mouse IRDye 800CW antibody (1:10,000; LI-COR). TILRR protein bands (70 kDa, green color, cropped) are shown on the top of the bar graph (see Figure S2A for un-cropped blot image). Untransferred proteins in the gel after iBlot transfer were stained with Coomassie blue staining (G-250, Bio-Rad), and the protein bands (~70 kDa, red color, cropped) are shown on the top of the bar graph (see Figure S2B for un-cropped gel). The bar graph shows the TILRR protein intensity (R.F.U). (B) TILRR protein intensity (R.F.U) (median with IQR) of PBMC lysates observed in four study subjects (n=4) including humans, rhesus monkeys, and cynomolgus monkeys. (C) TILRR protein expression between humans and monkeys. Data are shown as median with IQR in figures B and C. A t-test with 95% CI was used for statistical comparisons and all p<0.05 were reported as statistically significant and presented with asterisks. The X-axis of figure A indicates the patients’ and monkeys’ identification numbers and Y-axis shows the signal intensity (R.F.U). kDa, kiloDalton; RFU, relative fluorescence units; n= number of subjects; ID#, identification number, Rhesus M, rhesus monkey; Cyno M, cynomolgus monkey.

Figure 4 TILRR protein expression in human cell lines. Cell line lysates (7 µg/lane protein) were examined by Western blot (green color, left) and Coomassie blue (red color, right). TILRR protein was probed by primary mouse anti-TILRR F218G4 mAb followed by secondary goat anti-mouse IRDye 800CW antibody (1:10,000; LI-COR). TILRR protein bands (70 kDa, green color, cropped) are shown on the top of the bar graph (Figure S3A shows the un-cropped blot image). Untransferred proteins in the gel after iBlot transfer were stained with Coomassie blue staining (G-250, Bio-Rad) and the protein bands (~70 kDa, red color, cropped) are shown on the top of the bar graph (Figure S3B shows the un-cropped gel). The bar graph shows the TILRR protein intensity (R.F.U) for each tissue lysate (n=1) where X-axis demonstrates human cell lines and Y-axis represents signal intensity (R.F.U). kDa, kiloDalton; RFU, relative fluorescence units.

Figure 5 Anti-TILRR mAb is specific for TILRR protein. Tissues lysates (2.5 µg/lane protein) (n=2) were examined by Western blot (green color, left) and Coomassie blue staining (red color, right). TILRR protein was probed by either primary mouse anti-TILRR F218G4 mAb or primary mouse IgG1 mAb (isotype control) (Abcam, Canada) followed by secondary goat anti-mouse IRDye 800CW antibody (1:10,000; LI-COR). TILRR protein bands (70 kDa, green color, cropped) are shown on the top of the bar graph (see Figure S4A for un-cropped blot image). Untransferred proteins in the gel after iBlot transfer were stained with Coomassie blue staining (G-250, Bio-Rad), and the protein bands (~70 kDa, red color, cropped) are shown on the top of the bar graph (see Figure S4B for un-cropped gel). The bar graph shows the TILRR protein intensity (R.F.U) for each tissue lysate where X-axis represents human tissue lysates and Y-axis indicates the signal intensity (R.F.U). kDa, kiloDalton; RFU, relative fluorescence units.