Fig. 1: calpain2a interacts with TRAF6 and inhibits the NF-κB Signaling Pathway.

a List of TRAF6 interactome based on Label-free quantification intensity (section). b, c MKC cells seeded in 6 cm² dishes. After 24 h, cell lysates were immunoprecipitated (IP) with anti-TRAF6 or anti-calpain2a affinity gels. Then the immunoprecipitates and cell lysates were analyzed by IB with the anti-TRAF6 and anti-calpain2a Abs, respectively. d, e HEK 293 cells seeded in 6 cm² dishes were transfected with the plasmids calpain2a-Flag and TRAF6-Myc (2 μg each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Myc d or anti-Flag e affinity gels. Then the immunoprecipitates and cell lysates were analyzed by IB with the anti-Myc and anti-Flag Abs, respectively. f The same amino acids among human calpain2a (Hu-calpain2), mouse calpain2a (Mu-calpain2), zebrafish calpain2a (ZF-calpain2a) and miiuy croaker calpain2a (M-calpain2a) are highlighted with black background. g–i EPC cells were transfected with calpain2a-Flag or empty vector together with the NF-κB, IL-1β, and IL-8 luciferase reporters. At 24 h post-transfection, cells were untreated (Mock) or treated with LPS for 6 h. The luciferase activity value was achieved against the Renilla luciferase activity (n = 3 per group). Western blot analysis was used to measure the expression of transiently transfected calpain2a-Flag. The expression of Tubulin was used as a loading control. Data were analyzed by two-way ANOVA (g, h, i). ^**p  <  0.01. All experiments were performed in at least three independent experiments.