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. 2014 Nov 14;65(1):124–133. doi: 10.1136/gutjnl-2014-307545

Figure 6.

Figure 6

The C-terminus of STK33 binds directly to c-Myc. (A) Endogenous STK33 bound to c-Myc in hepatocellular carcinoma (HCC) samples. Endogenous STK33 was immunoprecipitated from the HCC tumour lysates using mouse STK33 antibody, followed by immunoblotting with antibodies to STK33 and c-Myc. Mouse IgG (mIgG) was used as an immunoprecipitation-negative control antibody. Sample loading controls are shown (2% input). (B) Endogenous STK33 bound to c-Myc in HCC samples. Endogenous c-Myc was immunoprecipitated from the HCC tumour lysates using rabbit c-Myc antibody, followed by immunoblotting with antibodies to c-Myc and STK33. Rabbit IgG (rIgG) was used as an immunoprecipitation-negative control antibody. Sample loading controls are shown (2% input). (C) Colocalisation of STK33 and c-Myc by immunofluorescent staining with STK33 antibody and c-Myc antibody in human HCC samples (upper) and diethylnitrosamine (DEN)-induce mouse liver tumour samples (lower). DAPI (4’,6-diamidino-2-phenylindole; blue) was used to indicate the nucleus. (D) STK33 binds to c-Myc in HKCL and SMMC7721 cells. STK33 was immunoprecipitated from the cell lysates followed by immunoblotting using antibodies to STK33 and c-Myc. mIgG was used as an immunoprecipitation-negative control antibody. (E) Scheme for c-Myc and STK33 mutants. (F) STK33 directly binding to c-Myc. Isolated His-fused c-myc protein (His-c-Myc), N-terminus of c-Myc (His-c-Myc-N), C-terminus of c-Myc (His-c-Myc-C), and GST-fused STK33 protein (GST-STK33) were incubated with glutathione beads as indicated. After extensive washing, they were immunoblotted with the His antibody (α-His); GST-STK33 was detected with GST antibody (α-GST). Sample loading controls are shown (2% input). (G) c-Myc bound to C-terminus of STK33. Isolated His-fused c-myc protein (His-c-Myc), GST-fused N-terminus of STK33 (GST-STK33-N) and C-terminus of STK33 (GST-STK33-C) were incubated with glutathione beads as indicated. After extensive washing, they were immunoblotted with the His antibody (α-His); GST-STK33 was detected with GST antibody (α-GST). Sample loading controls are shown (2% input). (H) C-terminus of STK33 blocked endogenous STK33/c-Myc association. c-Myc antibodies were used to immunoprecipitate c-Myc from SMMC7721 cell lysates, STK33 antibodies were used to detected endogenous STK33, and Flag antibodies were used to detect STK33-N or STK33-C.