Figure 4.

4A. Pre-incubation of glomeruli with CLCF1-CRLF1 heterodimer did not block the effect of TNFα or IL6. Isolated rat glomeruli were pre-incubated with CLCF1-CRLF1 (5–20 ng/mL) for 15 minutes followed by addition of TNFa (10ng/mL) or IL6 (1 ng/mL) and further incubation for 15 minutes at 37°C. TNFα and IL6 each caused a significant increase in P_alb compared to control (*, P<0.001 vs. control). CLCF1-CRLF1 did not block the increase in P_alb caused by TNFα or IL6. N=15 glomeruli from 3 rats (5 glomeruli from each rat) in each group.

4B and 4C. CLCF1-CRLF1 heterodimer blocked the CLCF1- induced phosphorylation of STAT3. Figure 4B shows a representative Western blot image to demonstrate the upregulation of STAT3 (Tyr705) phosphorylation by CLCF1 and the blocking effect of CLCF1-CRLF1 heterodimer. Control group represents untreated glomeruli. Glomeruli were incubated with CLCF1 (10ng/mL) for 15 minutes in one group. In additional groups, glomeruli were pre-incubated with heterodimer CLCF1-CRLF1 (10–40ng/mL) for 15 minutes followed by addition of CLCF1 (10ng/mL) and incubation for 15 minutes at 37°C. Thus, the ratio of CLCF1 to the heterodimer CLCF1-CRLF1 ranged from 1:1 to 1:4 (ng:ng) or a molar ratio of approximately 1:0.3 to 1:1.25. Total protein lysates were resolved by SDS-PAGE followed by Western blotting using anti-pSTAT3 (Tyr705) as the primary antibody.

Figure 4C. The bar graph shows results of quantitative analysis of protein band intensities. Changes in STAT3 phosphorylation (Tyr705) were determined by semi-quantitative image analysis. Background subtracted intensities were normalized by the loading control β-actin. Ratios of intensities (Experimental/control) are presented in the bar graph. CLCF1 caused significant increase in pSTAT3 (Tyr705) (*, P<0.001 control vs. CLCF1 alone). CLCF1-induced increase in pSTAT3 (Tyr705) was attenuated by pretreatment with 30 or 40 ng/mL CLCF1-CRLF1 (*, P<0.001 vs. CLCF1 alone). Mean±SEM of three separate experiments are shown.