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. 2019 Aug 1;9(8):1722–1733.

Figure 3.

Figure 3

USP44 stabilizes FBP1 in pancreatic cancer. (A) 293T cells were transfected with Flag-FBP1 and Myc-USP44 for 24 h. Whole-cell lysate (WCL) were collected for Western blot analysis of reciprocal co-immunoprecipitation (co-IP). (B) The WCL of PANC-1 were used for Western blot analysis of reciprocal co-immunoprecipitation of endogenous FBP1 and USP44 proteins. (C) Schematic diagram depicting a set of GST-FBP1 recombinant protein constructs. Western blot analysis of USP44 proteins in PANC-1 pulled down by GST or GST-FBP1 recombinant proteins. (D and E) BxPC-3 cells were infected with indicated shRNAs for 72 h. The cells were collected for Western blot analysis (C) and RT-qPCR analysis (D). The data are presented as the mean values ± SD (n = 3). n.s., not significant. (F and G) PANC-1 cells were transfected with indicated plasmids for 48 h. The cells were collected for Western blot analysis (E) and RT-qPCR analysis (F). The data are presented as the mean values ± SD (n = 3). n.s., not significant. (H) PANC-1 cells were infected with indicated shRNAs for 72 h. 50 μg/μl of cycloheximide (CHX) was added to the culture-medium of PANC-1 cells. The WCL of cells were collected for Western blot analysis at different time points as indicated. The protein expression level of FBP1 was normalized to the protein level of GAPDH and then to the value at the 0 h time point. (I) PANC-1 cells transfected with the indicated plasmids for 24 h. The cells were treated with 20 μM of MG132 for 8 h before harvest for Western blotting analysis. (J) PANC-1 cells transfected with the indicated plasmids for 24 h. The cells were treated with 20 μM of MG132 for 8 h before harvest for Western blotting analysis. (K) Purified FBP1, and USP44 were incubated for 2 h and the reaction was harvested for Western blotting analysis. (L) Western blot analysis of WCL after PANC-1 cells were transfected with indicated constructs for 24 h. (M) PANC-1 cells transfected with the indicated constructs for 48 h. The cells were treated with 20 μM of MG132 for 8 h before harvest for Western blotting analysis.