Fig. 4

LDHA is targeted for lysosomal degradation by binding to SQSTM1. a Flag-LDHA is stably expressed in AGS or HGC27 cells after transfection. b and c LDHA is degraded by the lysosomal pathway. Flag-LDHA was transfected into AGS or HGC27 cells for 24 h, then the cells were treated with BafA1 (20 nM) (b) or chloroquine (50 μM) (c) for the indicated time points. The cell lysates were collected and analyzed by western blot. LDHA binds to SQSTM1. d Flag-LDHA interacts with His-SQSTM1 in AGS and 293 T cells. The cells were co-transfected with Flag-LDHA and His-SQSTM1. For the co-IP experiments, anti-FLAG antibody was used for pulldown and anti-His antibody for western blot. e PLA assay for detection of LDHA and SQSTM1 was performed in AGS cells treated with or without BafA1 (20 nM). f Colocalization of LDHA and SQSTM1 in AGS cells treated with or without BafA1 (20 nM) was observed by confocal microscopy. Magnification: 800x. The degree of colocalization was assessed using the Pearson’s coefficient (g) and the Mander’s coefficients (h). Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001