Fig. 5: calpain2a inhibits TRAF6 ubiquitin-ligase activity.
From: The protease calpain2a limits innate immunity by targeting TRAF6 in teleost fish
a Ubiquitination of endogenous TRAF6 in MKC cells transduced with calpain2a-Flag and calpain2a-ΔCysPc-Flag and unchallenged (−) or challenged with LPS (+), assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-TRAF6 and input immunoblot analysis with indicated Abs. b, c HEK293 cells were cotransfected with TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. d, e HEK293 cells were cotransfected with TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag (0.5 μg, 1 μg), calpain2a-ΔCysPc-Flag (0.5 μg, 1 μg) or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. f Ubiquitination of overexpressed TRAF6 in MKC cells transduced with calpain2a-ΔCysPc-Flag, assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-Myc and input immunoblot analysis with indicated Abs. g, h HEK293 cells were cotransfected with TAK1-Myc, TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. All the immunoprecipitates and input immunoblot analysis with anti-Myc, anti-Flag, anti-HA, and anti-Tubulin Abs. All experiments were performed in at least three independent experiments.
