Fig. 3.

RNF144A targets oncoprotein HSPA2 for proteasome-dependent degradation. a Cells stably expressing pCDH and Flag-RNF144A were subjected to immunoblotting analysis with the indicated antibodies. Vinculin was used as loading control. b MCF-7 and SK-BR-3 cells stably expressing pCDH and RNF144A were treated with or without 10 μM MG-132 for 6 h as indicated in Supplementary Fig. S4B and then subjected to iTRAQ-based quantitative proteomics. Quantitative results of HSPA2 protein levels are representative of the mean value of two independent experiments. c HEK293T cells were transfected with HA-HSPA2 expression vector alone or in combination with increasing doses of Flag-RNF144A plasmid DNA. After 48 h of transfection, lysates were subjected to immunoblotting with the indicated antibodies. d MDA-MB-231 cells stably expressing pCDH and Flag-RNF144A were subjected to immunoblotting analysis with the indicated antibodies. e Immunoblotting analysis of RNF144A and HSPA2 in the indicated breast cancer cell lines. f MDA-MB-231 cells were treated with 10 μM of MG-132 for the indicated time points and subjected to immunoblotting analysis with the indicated antibodies. g MDA-MB-231 cells stably expressing pCDH and Flag-RNF144A were treated with DMSO or 10 μM of MG-132 for 6 h and then subjected to immunoblotting analysis with the indicated antibodies. h, i Lysates from stable MCF-7 and MDA-MB-231 cells expressing shNC and shRNF144A were subjected to immunoblotting analysis with the indicated antibodies. j, k qPCR analysis of HSPA2 mRNA levels in MCF-7 and MDA-MB-231 cells stably expressing shNC and shRNF144A. l MCF-7 cells stably expressing shNC and shRNF144A were treated with 100 μg/ml of CHX for the indicated times and then subjected to immunoblotting with the indicated antibodies. Quantitative results of HSPA2 protein levels (HSPA2/vinculin) are representative of the mean value of two independent experiments and are shown in the lower panel