Figure 3.

ZFP91 K48-ubiquitinates and degrades hnRNP A1. (A) Proteins that interacted with ZFP91 were identified by co-IP and mass spectrometry assays using HEK293T cells as a cell model. (B, C) ZFP91-Flag and hnRNP A1-HA plasmids were transfected into HCC cells HCC MHCC-LM3 (B) and SK-hep1 (C), ZFP91-Flag (B) and hnRNP A1-HA (C) complexes were co-immunoprecipitated with anti-Flag and HA antibodies, and hnRNP A1 and ZFP91 were detected, respectively. (D) MHCC-LM3 HCC cells were transfected with ZFP91-Flag plasmid, hnRNP A1 protein level was determined. (E) SK-hep1 HCC cells were transfected with anti-ZFP91 siRNA, hnRNP A1 protein level was determined. (F) ZFP91 and hnRNP A1 protein levels in six pairs of primary HCC tissues (T) and corresponding N tissues used in Figure 1B were detected. The correlations of ZFP91 with hnRNP A1 were analyzed in the right panel. (G) MHCC-LM3 cells transfected with ZFP91-Flag plasmids for 36 h were incubated with cycloheximide (CHX) for the indicated times. The indicated proteins were analyzed (left panel), and the relative hnRNP A1 protein level is illustrated graphically (right panel). (H) MHCC-LM3 cells were cotransfected with the indicated plasmids for 36 h, followed by treatment with 10 µM MG132; the polyubiquitination level of hnRNP A1 was detected. (I) SK-hep1 cells were cotransfected with the indicated plasmids together with anti-ZFP91 siRNA for 36 h, followed by treatment with MG132; the polyubiquitination level of hnRNP A1 was detected. (J) MHCC-LM3 cells were cotransfected with wild type HA-ub or its mutants together with ZFP91-HA and hnRNP A1-Flag plasmids for 36 h, followed by treatment with MG132; the polyubiquitination level of hnRNP A1 was detected.