Fig. 4

N-CoR was a substrate of chaperone mediated autophagy and modulated by LAMP-2A siRNA. a The association between N-CoR and LAMP-2A (left), N-CoR and Hsc70 (right) was confirmed by co-immunoprecipitation assay in U87-MG cells. The cell lysates immunoprecipitated by either LAMP-2A antibody or Hsc70 displayed positive N-CoR protein expression as detected by N-CoR antibody. b Distribution of LAMP-2A (green) and N-CoR (red) in U87-MG cells was demonstrated in immunofluorescence analysis. DNA (blue signal) was stained with DAPI. Significant co-localization between LAMP-2A and N-CoR was observed in vehicle and control siRNA (CT-siR) groups. After treatment with LAMP-2A siRNA (LAMP-2A siR), remarkable cytoplasmic and intra-nucleus accumulation of N-CoR was observed as compared with vehicle and CT-siR groups. c Nuclear and cytoplasmic proteins were separated by NE-PER™ kit. WB analysis showed that cytoplasmic LAMP-2A expression was inhibited, and N-CoR in both cytoplasmic and nuclear compartments were accumulated upon treatment with LAMP-2A siRNA as compared with CT-siR treatment. d the protein expression of N-CoR was significantly increased after LAMP-2A knockdown as compared with CT-siR in U87-MG cells, while the mRNA level of N-CoR remained unchanged. e Immunohistochemistry analysis confirmed increased protein level of N-CoR (brown signal) after LAMP-2A was inhibited in U87-MG cells. Nucleus (blue signal) was stained with hematoxylin. f Compared with normal control (NC), starvation overnight resulted in activation of CMA pathway, as demonstrated by increased protein expression of LAMP-2A, and significantly decreased N-CoR level. The data are mean ± SEM from three independent experiments. Protein and mRNA levels are expressed relative to vehicle treatment group set as 1. Significant changes are set as p < 0.05 and represented by asterisk (One-Way ANOVA; Bonferroni's test)