Fig. 4: calpain2a inhibits TRAF6 protein expression.

a The time gradient experiment of empty vectors or calpain2a-Myc plasmids together with TRAF6-Flag was conducted in EPC cells. The cell lysates were subjected to IB with anti-Myc, anti-Flag, and anti–Tubulin Abs. RNA was extracted from cells and reverse transcribed, then TRAF6 and actin were amplified by PCR primers. b EPC cells were seeded in 12-well plates overnight and co-transfected with TRAF6-Flag and calpain2a-Myc (0.3, 0.6, or 0.9 μg) for 48 h. The expression of TRAF6-Flag and calpain2a-Myc proteins were detected by Western blotting. RNA was extracted from cells and reverse transcribed, then TRAF6 and actin were amplified by PCR primers. c calpain2a-Myc or empty vectors were co-transfected with TRAF6-Flag into EPC cells. At 36 h post-transfection, the transfected cells were treated with cycloheximide (CHX) for 2 or 4 h. d MKC cells were transfected with calpain2a-Flag or empty vectors. At 48 h post-transfection, the cell lysates were subjected to IB with anti-TRAF6, anti-Flag, and anti-Tubulin Abs. e, f EPC cells were transfected with the indicated plasmids in the presence or absence of MG132, 3-MA, NH₄CL, E-64 (50 or 75 μM), or PMSF for 6 h before immunoblot analysis was performed. g MKC cells were transfected with calpain2a-Flag in the presence or absence of E-64 (50 or 75 μM) for 6 h before immunoblot analysis was performed. h calpain2a-Flag and calpain2a-ΔCysPc-Flag were co-transfected with TRAF6-HA into EPC cells. At 48 h post-transfection, the cell lysates were subjected to IB with indicated Abs. i calpain2a-Flag and calpain2a-ΔCysPc-Flag were co-transfected into MKC cells. At 48 h post-transfection, the cell lysates were subjected to IB with anti-TRAF6, anti-Flag, and anti-Tubulin Abs. j IL-1β, IL-8 mRNA in MKC stably transduced with calpain2a-Flag and calpain2a-ΔCysPc-Flag and treated with saline (0) or challenged with LPS for 4 h (n = 3 per group). Relative mRNA level was normalized to the expression of the gene encoding β-actin in each sample. All experiments were performed in at least three independent experiments. Data were analyzed by two-way ANOVA (j). ^**p  <  0.01.