Fig. 3: STAMBP deubiquitinates and stabilizes RAI14. | Experimental & Molecular Medicine

Fig. 3: STAMBP deubiquitinates and stabilizes RAI14.

From: The deubiquitinating enzyme STAMBP is a newly discovered driver of triple-negative breast cancer progression that maintains RAI14 protein stability

Fig. 3

a qPCR analysis of STAMBP and RAI14 gene expression in MDA-MB-231 and BT549 cells stably transfected with negative control (NC) or STAMBP shRNA. The mean ± SD (n = 3), *p < 0.05. b MDA-MB-231 and BT549 cells transiently transfected with STAMBP shRNA #1 or control shRNA were treated with cycloheximide (CHX, 100 μg/ml) for the indicated time periods. The protein levels of STAMBP and RAI14 were measured by Western blotting. The intensity of RAI14 expression for each time point was quantified by ImageJ software. The mean ± SD (n = 3). *p < 0.05. c MDA-MB-231 and BT549 cells were treated with MG132 (10 μM) or bafilomycin A1 (0.1 μM) for the indicated time. The protein levels of RAI14 were measured by Western blotting. GAPDH was used as a loading control. d MDA-MB-231 cells were treated with or without MG132 for 6 h. The extracts were immunoprecipitated with anti-RAI14 antibodies and immunoblotted with anti-ubiquitin, anti-K48-linked ubiquitin, and anti-RAI14 antibodies. e MDA-MB-231 cells stably transfected with STAMBP shRNA #1 or control shRNA were treated with or without MG132 for 12 h. The expression of RAI14 and STAMBP was assessed. f MDA-MB-231 cells stably transfected with two individual STAMBP shRNAs or control shRNA were treated with MG132 for 6 h. The extracts were immunoprecipitated with anti-RAI14 antibodies and immunoblotted with anti-ubiquitin, anti-K48-linked ubiquitin, and anti-RAI14 antibodies. g Immunoblotting of STAMBP and RAI14 in MDA-MB-231 and BT549 cells stably expressing vector or Flag-STAMBP. h MDA-MB-231 cells stably expressing vector or Flag-STAMBP were treated with MG132 for 6 h. The extracts were immunoprecipitated with anti-RAI14 antibodies and immunoblotted with anti-ubiquitin, anti-K48-linked ubiquitin, and anti-RAI14 antibodies. i MDA-MB-231 cells stably expressing STAMBP shRNAs and/or STAMBP expression plasmids were treated with MG132 for 6 h. The extracts were immunoprecipitated with an anti-RAI14 antibody and immunoblotted with the indicated antibody.

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