Fig. 3: PSMD14 deubiquitinates CARM1. | Cell Death & Disease

Fig. 3: PSMD14 deubiquitinates CARM1.

From: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

Fig. 3

A Control and PSMD14-knockdown Huh7 cells were treated with MG132 at 10 μM for 6 h. The cell lysates were immunoprecipitated with an anti-CARM1 antibody, and the immunocomplexes were immunoblotted with anti-CARM1 and anti-ubiquitin antibodies. B Huh7 cells were pretreated with 10 μM capzimin for 24 h, and cells treated with equal amounts of DMSO served as controls. All cells were treated with MG132 at 10 μM for 6 h. The cell lysates were then immunoprecipitated with anti-CARM1 antibodies, and the immunocomplexes were immunoblotted with anti-CARM1 and anti-ubiquitin antibodies. C FLAG-CARM1 and HA-Ubi were transiently transferred into HEK293T cells expressing GFP-PSMD14-WT or GFP-PSMD14-MUT. Cells were then treated with MG132 (10 μM) for 6 h. The cell lysates were immunoprecipitated with an anti-FLAG M2 affinity gel. The ubiquitination levels of CARM1 were detected using anti-HA antibodies. D FLAG-CARM1 and HA-Ubi were transiently transferred into HEK293T cells, which were subsequently purified with FLAG antibodies and protein G beads. Moreover, GFP-PSMD14-WT and GFP-PSMD14-MUT were separately transferred into another two sets of HEK293T cells and purified with GFP antibodies and protein G beads. The purified Ubi-FLAG-CARM1 and GFP-PSMD14 proteins were incubated for 1 h. Then, FLAG-CARM1 was immunoprecipitated with anti-FLAG M2 affinity beads and detected with the indicated antibodies. E HEK293T cells were transfected with the indicated plasmids and treated with MG132 (10 μM) for 6 h. The cell lysates were immunoprecipitated with anti-FLAG M2 affinity beads. The ubiquitination levels of CARM1 were detected using anti-HA antibodies. F Schematic illustration of CARM1 truncation plasmids and lysine sites predicted to be modified by ubiquitination. G HEK293T cells expressing GFP-PSMD14 were transfected with FLAG-tagged full-length or truncated CARM1. The cell lysates were immunoprecipitated with anti-GFP antibodies. H HEK293T cells were transfected with the indicated plasmids and treated with MG132 (10 μM) for 6 h. The cell lysates were immunoprecipitated with an anti-FLAG M2 affinity gel. The ubiquitination levels of CARM1 were detected using anti-HA antibodies.

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