Fig. 7

SQSTM1/P62 interacts with TPI1 to down-regulate TPI1 protein level in BRCA. A Silver staining assay identified TPI1-specific bands. B Mass spectrometry identified 3 mainly interacting proteins. C GSEA analysis showed TPI1 involved in ubiquitination degradation. D IP analysis of MDA-MB-231 and T47D cells with antibodies against TPI1 and P62. P62 and TPI1 were analyzed by immunoblotting. E Western blot and qRT-PCR used to quantify expression of TPI1 and P62 in MDA-MB-231 cells. Overexpression of P62 down-regulated TPI1 at protein level, but not at mRNA level. NS represents no statistical significance. F MDA-MB-231 cells were transfected with P62 plasmid and treated with or without MG132 (10 μM) for 6 h. Cell lysates were analyzed by western blot with indicated antibodies. G Ubiquitination assay to examine effects of P62 on TPI1 ubiquitination. Flag-Ub and p62 plasmid were co-transfected into MDA-MB-231 cells, then incubated with MG132 for 6 h