Fig. 3

LINRIS is involved in the autophagic degradation of IGF2BP2. a Schematic structures of IGF2BP2 proteins and three truncated mutants (T1: 1–193; T2: 157–427; T3: 341–913) of IGF2BP variants used in this study. Orange boxes are RRM domains, and blue boxes are KH domains. b RIP assays were performed using anti-FLAG antibodies and HCT116 cells transfected with vectors expressing the FLAG-tagged FL and the truncated mutants (T1-T3) of IGF2BP2. The data shown represent three independent experiments. The data are shown as the mean ± SD; n = 3 independent experiments, two-tailed Student’s t-test, *P < 0.05, **P < 0.01. c IP assays were performed using anti-FLAG antibodies and HCT116 cells transfected with vectors expressing the FLAG-tagged WT or IGF2BP2 mutants (K77R and K139R). d Western blot analysis following RNA pull-down assays shows that the K139R mutation of IGF2BP2 blocked its binding to LINRIS in CRC cells. GAPDH was used as the loading control. e CRC cells with or without shRNAs specific for LINRIS were treated with 20 μg/ml CHX or a vehicle for the indicated periods of time. IGF2BP2 levels were analyzed by western blotting. f CRC cells with LINRIS knockdown and control cells were treated with or without MG132 (5 μM) for 12 h. Cell lysates were analyzed by Western blotting with GAPDH as the loading control. g Illustration of the protein degradation process via two main pathways. h CRC cells with LINRIS knockdown were treated with or without Baf A1 (100 nM, 24 h) and NH₄Cl (10 mM, 4 h). Cell lysates were analyzed by Western blotting with GAPDH as the loading control. i Western blotting shows the levels of IGF2BP2 in the indicated cells after treatment with or without EBSS for 3–6 h or Rap (100 nM) for 24 h. GAPDH was used as the loading control. j Confocal microscopy of HCT116 cells treated with or without EBSS for 3 h. Scale bar, 50 μm