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. 2021 Feb 24;40(11):2112–2129. doi: 10.1038/s41388-021-01679-8

Fig. 5. USP13 interacts with and deubiquitinates Mcl-1 in cervical cancer cells.

Fig. 5

A HEK293T cells were transfected with V5-Mcl-1, Flag-USP13, or both V5-Mcl-1 and FLAG-USP13. Cells were treated with 10 µM MG132 for 6 h and either Mcl-1 or USP13 were immunoprecipitated using an anti-V5 or anti-FLAG antibody. Co-immunoprecipitated Mcl-1 or FLAG-USP13 were detected using the respective antibodies. GAPDH was used as a loading control. B Endogenous Mcl-1 and USP13 were immunoprecipitated from HeLa and SiHa cells after treatment with 10 µM MG132 using an anti-Mcl-1 or anti-USP13 antibodies. Co-immunoprecipitated Mcl-1 or USP13 were detected using the respective antibodies. GAPDH was used as a loading control. C HEK293T cells were co-transfected with V5-Mcl-1 and HA-Ubiquitin, with or without Flag-USP13 or a FLAG-USP13 mutant (C345A). Cells were treated with 10 µM MG132 for 6 h and V5-Mcl-1 was immunoprecipitated using an anti-Mcl-1 antibody. Ubiquitinated Mcl-1 was detected using an anti-HA antibody. D HEK293T cells were co-transfected with V5-Mcl-1, HA-Ubiquitin or mutant Ubiquitin (K48R or K63R), with or without Flag-USP13. Cells were treated with 10 µM MG132 for 6 h and V5-Mcl-1 was immunoprecipitated using an anti-V5 antibody. Ubiquitinated Mcl-1 was detected using an anti-HA antibody. E HeLa and SiHa cells were transfected with a pool of four specific USP13 siRNA for 72 h. Cells were treated with 10 µM MG132 for 6 h before harvesting. Mcl-1 was immunoprecipitated using an anti-Mcl-1 antibody. Ubiquitinated Mcl-1 was detected using an anti-ubiquitin antibody. GAPDH was used as a loading control.