Fig. 4.

RNF144A interacts with HSPA2 and promotes its polyubiquitination. a HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, total cellular lysates were subjected to IP and immunoblotting analysis with the indicated antibodies. b HEK293T cells were transfected with the indicated expression vectors and indirect IF staining was carried out after 48 h of transfection with the indicated antibodies. DAPI was used as a nuclear staining. c MCF-7 and MDA-MB-231 cells were treated with 10 μM MG-132 for 6 h to enhance basal levels of endogenous RNF144A and HSPA2. Then, total cellular lysates were subjected to IP analysis with control IgG or an anti-HSPA2 antibody, followed by immunoblotting with the indicated antibodies. d Lysates from MCF-7 cells were incubated with purified GST or GST-RNF144A protein and then subjected to immunoblotting analysis with the indicated antibodies. e, f HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were incubated with 10 μM of MG-132 for 6 h and then subjected to sequential IP and immunoblotting analysis with the indicated antibodies. g HEK293T and MCF-7 cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were subjected to immunoblotting analysis with the indicated antibodies. h, i HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were treated with 100 μg/ml of CHX for the indicated times and then subjected to immunoblotting with the indicated antibodies (h). Quantitative results of HSPA2 protein levels (HSPA2/vinculin) are representative of the mean value of two independent experiments (i)