Figure 5.

Integrins activate NF-κB transcription for cIAP1 expression in cardiomyocytes in vitro. Collagen/RGD model was conducted as in Fig. 1B. A) Feline cardiomyocytes were stimulated with 9 mM RGD, and Triton-insoluble and soluble samples were used for Western blots with anti-ubiquitin, anti-IκB, and anti-pIκB (S32/S36) antibodies and normalized to actin and GAPDH, respectively. Cell experiments were done in quadruplicate. B) Feline cardiomyocytes were infected with Ad-NFκB-luc (MOI 10) for 36 h prior to collagen layering ± 9 mM RGD for 2 h. Total RNA was subjected to real-time RT-PCR for luciferase expression. 18 S mRNA was used as an internal control. Summary graph represents average of 3 experiments; error bars = se. *P < 0.05 vs. infected control. C) Feline cardiomyocytes were infected with either Ad-β-Gal (MOI 150) or Ad-IκB-S32A (MOI 150) for 36 h before stimulating with collagen containing 9 mM RGD for 1 h. Western blots were performed on insoluble and soluble fractions with anti-ubiquitin, anti-IκB, and anti-pIκB (S32/S36) antibodies; loading was normalized to actin and GAPDH, respectively. D) Murine cardiomyocytes were infected with either Ad-β-Gal (MOI 150) or Ad-IκB-S32A (MOI 150) for 36 h before stimulating with 6 mM RGD in collagen for 1 h. Total RNA was extracted and subjected to real-time RT-PCR for cIAP1. Endogenous GAPDH mRNA was used as an internal control. Summary graph represents average of 3 three experiments; error bars = se. *P = 0.01 vs. β-Gal control; #P = 0.005 vs. β-Gal + RGD.