Fig 1: Loss of DIS3L2 expression upregulates AZGP1 mRNA and protein levels and inhibits the mTOR signaling pathway. A AZGP1 mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control) mRNA and determined by RNA-seq (left) and RT-qPCR (right) in SW480 cells treated with siRNAs targeting LUC (control condition), DIS3L2 or DIS3L2 + TUTs. B mRNA stability of AZGP1 in SW480 cells transfected with siRNAs targeting LUC (control condition) and DIS3L2. The mRNA levels were determined by RT-qPCR at various time points (0, 60, 120, 240 min) after DRB treatment. AZGP1 mRNA decay rates were plotted by normalizing the mRNA level of each time point to that of 0 h in each condition; statistical significance is relative to mock condition at each time point. C Bar plot representing AZGP1 protein levels determined by Western blot in siLUC- and siDIS3L2-treated SW480 cells. On the right, a representative Western blot analysis for DIS3L2, AZGP1 and a–tubulin (loading control). D Bar plots representing Cyclin D1 and p/t-mTOR protein levels determined by Western blot in SW480 depleted from DIS3L2 or DIS3L2 + TUTs, after normalizing to protein levels of siLUC-treated cells. Representative Western blot analysis for phospho-Ser2448-mTOR (p-mTOR), total-mTOR (t-mTOR), TUT7, DIS3L2, AZGP1, Cyclin D1 and a–tubulin (loading control) to monitor the impact of DIS3L2 depletion in the mTOR signaling pathway in SW480 cells. E Bar plots representing p/t-mTOR and p/t-4EBP protein levels determined by Western blot in SW480 cells treated with siRNAs targeting LUC (control condition) or DIS3L2 with or without DIS3L2 overexpression. Representative Western blot analysis for p-mTOR, t-mTOR, DIS3L2, phosphor-Ser-112-4EBP (p-4EBP), total-4EBP (t-4EBP) and a-tubulin. n = 3, statistical significance relative to mock condition are indicated as: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001
Supplier Page from MilliporeSigma for Anti-AZGP1 antibody produced in rabbit