Fig. 3

K222 succinylation does not affect the ubiquitination of LDHA. a The expression of LDHA in different cell lines. b and c LDHA is not degraded via the ubiquitin-proteasome pathway. Flag-LDHA was transfected into AGS cells for 24 h, then the cells were treated with CHX (10 μg/ml) (b) or MG132 (10 mM) (c) for the indicated time points. The cell lysates were collected and analyzed by western blot. d LDHA is mainly ubiquitinated on K63. AGS cells were transfected with the indicated plasmids and the IP was performed to pull down Flag-LDHA. The ubiquitination was detected by testing HA expression. e-g K222 mutation does not affect the ubiquitination of LDHA. AGS cells were transfected with indicated plasmids and the IP was performed to pull down Flag fusion proteins. The modification by ubiquitin (e), K63- (f) or K48- (g) ubiquitin was determined by HA antibody staining. h K222suc-LDHA is ubiquitinated and mainly modified by K63-ubiquitin. AGS cells were transfected with indicated plasmids and the co-IP was performed to pull down Flag-K222E protein. The ubiquitination was detected by measuring HA expression. Data are presented as mean ± SEM, *P < 0.05, ***P < 0.001