Fig. 5

K222suc can decrease the binding of K63-ubiquitinated LDHA to SQSTM1, thereby reducing its degradation. a Flag-K222E (upper) or Flag-K222R (lower) fusion protein is stably expressed in AGS cells after transfection. b-e Flag-K222E protein was accumulated in cells and K222suc could inhibit the lysosomal degradation of LDHA. After transfection with Flag-K222E or Flag-K222R plasmid for 24 h, AGS cells were treated with CHX (10 μg/ml) (b), MG132 (10 mM) (c), chloroquine (50 μM) (d), or BafA1 (20 nM) (e). The cell lysates were collected at the indicated time points, and the K222E (upper) or Flag-K222R (lower) fusion protein was detected using anti-Flag antibody. f Flag-K222E binds to SQSTM1. g Succinylation of K222 decreases the binding of LDHA to SQSTM1. AGS cells were transfected with indicated plasmids, LDHA fusion proteins were immunoprecipitated by Flag antibody, and SQSTM1 protein was detected using anti-His antibody. h and i K63, rather than K48, ubiquitination promotes the interaction between LDHA and SQSTM1. AGS cells were transfected with Flag-LDHA and His-SQSTM1 with or without HA-K63 (H) or HA-K48 (I). Flag-LDHA was pulled down, and His-SQSTM1 was detected by western blot. j and k Neither K63 (j) nor K48 (k) ubiquitination can promote the interaction between Flag-K222E and SQSTM1. AGS cells were transfected with the indicated plasmids. Subsequently, Flag-K222E was immunoprecipitated, and His-SQSTM1 was detected by western blot. l and m K63 ubiquitination promotes the binding of LDHA to SQSTM1 leading to LDHA degradation. AGS cells were transfected with indicated plasmids for 24 h. Then, the cell lysates were collected at various time points, and Flag-LDHA was detected by western blot in the presence of K63-ubiquitin (l) or control GFP (m). n and o K63-linked ubiquitination did not promote the degradation of Flag-K222E. The experiments were performed as described in (l, m) but the Flag-K222E plasmid was used instead of Flag-LDHA. p BafA1 inhibited LDHA degradation promoted by K63 ubiquitination. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001