Fig. 5.

ZFP91 destabilizes TSPYL2 via promoting ubiquitin-mediated degradation. A.TSPYL2 mRNA expression in PANC-1 and BxPC-3 cells 48 h after lentiviral mediated ZFP91 knockdown. B–C. Relative ZFP91 and TSPYL2 protein expression in PANC-1 and BxPC-3 cells 48 h after lentiviral mediated ZFP91 knockdown B) or treatment with 50 or 200 μM ginsenoside Rg3 (C). For MG132+ group (down), MG132 (10 μM) was added 6 h before harvesting and western blotting assay. D-H. Cycloheximide pulse-chase assay was performed in PANC-1 cells with Flag-TSPYL2 overexpression alone or in combination with ZFP91 knockdown (D–E), ZFP91 overexpression (D and F) or ginsenoside Rg3 treatment (50 or 200 μM, 48 h) (G–H). 36 h after lentiviral infection, cells were treated with 10 μM CHX for the indicated time, followed by Western blot analysis. Representative images were presented (D and G) and the relative TSPYL2 protein levels were illustrated graphically (E, F and H). ^#, comparison between 0 and 50 μM groups; ∗, comparison between 0 and 200 μM groups. I-J. PANC-1 cells were coinfected with the indicated lentiviruses (HA-Ub, Flag-TSPYL2, and ZFP91 shRNA or Myc-ZFP91) for 36 h with or without the presence of ginsenoside Rg3 (Res.) (50 or 200 μM), followed by treatment with MG132 (10 μM, 6 h). Then, cell lysates were immunoprecipitated with an anti-Flag antibody. Ubiquitinated TSPYL2 was detected by Western blot assay with an anti-HA antibody. Gin.: ginsenoside Rg3.