Fig. 6

GSK3β promotes the binding and ubiquitination of IGF2BP2 by FBW7 in lung cancer cells. A A schematic diagram depicting GSK3β consensus motif in IGF2BP2 conserved region. B H1299 and A549 cells were harvested and immunoprecipitated with IgG, IGF2BP2, or GSK3β antibodies. C H1299 and A549 cells were transfected with EV or Myc-IGF2BP2 plasmids for 72 h. Cells were collected and immunoprecipitated with Myc antibody. D and E H1299 and A549 cells were transfected with indicated plasmids. After 48 h and 72 h, cells were harvested for RT-qPCR and Western blotting analyses. n = 3, one-way ANOVA. Data are presented as Mean ± SD. F A549 cells were transfected with Flag-GSK3β. After 48 h, the corresponding groups were treated with MG132 for another 12 h. Cells were harvested for Western blotting analysis. n = 3, one-way ANOVA. Data are presented as Mean ± SD. G-I A549 cells were transfected with indicated plasmids for 72 h or treated with GSK3β specific inhibitor TWS119 (10 μM) for 1 h. Cells were treated with Cycloheximide (CHX) and then collected at different time points for Western blotting analysis to detect IGF2BP2 levels. J and K A549 cells were transfected with indicated plasmids (Myc-IGF2BP2, Flag-GSK3β, shGSK3β, HA-Ub) for 48 h or treated with TWS119 (10 μM) for 1 h. Then cells were treated with MG132 for another 12 h. Cells were harvested for Western blotting analysis. L A549 cells were transfected with Flag-GSK3β, plasmid expressing wild-type IGF2BP2 (Myc-IGF2BP2-WT), and plasmid expressing IGF2BP2 with Thr306 and Thr310 residues mutated to alanine (Myc-IGF2BP2-AA) for 72h. Cells were collected and immunoprecipitated with Flag antibody for Western blotting analysis. M A549 cells were transfected with indicated plasmids (Myc-IGF2BP2-WT, Myc-IGF2BP2-AA, Flag-GSK3β, HA-Ub) for 48 h. Then cells were treated with MG132 for another 12 h. Cells were harvested for Western blotting analysis. N and O A549 cells were transfected with Flag-GSK3β for 48 h. Then cells were treated with MG132 for another 12 h. Cells were collected and immunoprecipitated with IgG, IGF2BP2, or FBW7 antibodies for Western blotting analyses. ns, not significant, P > 0.05; **, P < 0.01; ***, P < 0.001. Abbreviations: BP2, IGF2BP2