Fig. 5: NLRC5 interacts with CAVIN1 and regulates its stability.

a Immunoprecipitation of NLRC5 binding partners. NLRC5 was overexpressed in neonatal mouse cardiomyocytes (NMCMs) through Ad5-cTnT-NLRC5. The cell lysate was immunoprecipitated using either IgG (control) or anti-NLRC5 antibody. Immunoprecipitated samples were subjected to SDS-PAGE followed by silver staining. Co-immunoprecipitation (Co-IP) assays assessed the interaction between NLRC5 and CAVIN1 in both anti-NLRC5 pull-down NMCM samples (b) and anti-CAVIN1 pull-down NMCM samples (c). Input was used as positive controls and anti-IgG pull-down samples were assessed as negative controls. d Illustration of the truncated domains of CAVIN1. e Co-IP of HEK-293T cells overexpressing Flag-NLRC5 and HA-tagged CAVIN1 or truncated forms followed by Western blot. f Molecular docking analysis of the NLRC5 and CAVIN1 domains, with CAVIN1 in yellow and NLRC5 in cyan. g Expression of CAVIN1 and NLRC5 in NMCMs 48 h after transfected with Ad5-Ctrl or Ad5-cTnT-NLRC5 (n = 5 each). h Quantitative RT-PCR analysis of the mRNA levels of Cavin1 in NMCMs transfected with Ad5-Ctrl, Ad5-cTnT-NLRC5 or Ad5-cTnT-NLRC5i (n = 3 each). i Half-life of CAVIN1 protein in NMCMs treated with or without NLRC5 knockdown in the presence of 0.4mM CHX for 0, 4, 8, 12h (n = 3 each). j Western blot and corresponding quantification of CAVIN1 protein level in NMCMs treated with 0.4 mM CHX in the presence or absence of MG-132 (20 μM), bafilomycin A1 (100 nM), or E64d (10 μM) for 8 h (n = 4 each). k Western blot and corresponding quantification of CAVIN1 in NLRC5 overexpressed NMCMs with or without MG-132 (20 μM) for 8 h (n = 3 each). Data are presented as mean ± SD. g Standard unpaired Student t test. h, j, k 1-way ANOVA with Tukey multiple comparisons test. i Calculation using equation fitted by linear regression. *P < 0.05, **P < 0.01, and ***P < 0.001. ns indicates not significant.