Mouse anti human gapdh

Western blot was carried out as previously described^{27 (link)}. The primary antibodies used in Western blot were as follows: mouse anti-human Pontin (catalog SAB4200194; Sigma-aldrich, USA), mouse anti-human GAPDH (catalog BM3876; Boster, Wuhan, China), rabbit anti-human E2F1 (catalog 49286; Sabbiotech, USA), rabbit anti-human CDK1 (catalog 10762-1-AP; Proteintech, USA), rabbit anti-human CDK4 (catalog 12790 T; Cell Signaling Technology, USA), mouse anti-human cyclin B2 (catalog sc-28303; Santa Cruz Biotechnology, USA), mouse anti-flag tag (catalog F3165; Sigma-aldrich, USA). Co-IP was performed as previously described^{28 (link)}. In brief, cell extracts were immunoprecipitated with each 2 μg of indicated antibody (IgG, E2F1, or flag antibody) for 4–6 h and with protein A/G agarose beads (catalog No. sc-2003; Santa Cruz Biotechnology) overnight. Bound proteins were then washed, resuspended in protein sample buffer, separated by SDS-PAGE and detected by immunoblot.

Western blot was performed according to our previously described protocols [26 (link)]. The primary antibodies were as follows: mouse anti-human Pontin (catalogue SAB4200194; Sigma‒Aldrich), mouse anti-human GAPDH (catalogue BM3876; Boster, China), goat anti-human TGFβRII (catalogue AF-241-NA; Biotechne, USA), rabbit anti-human CTGF (catalogue A11067; Abclonal, China), rabbit anti-human SMAD2/SMAD3 (catalogue 5678; Cell Signaling Technology, USA), rabbit anti-human phospho-SMAD2/SMAD3 (catalogue 8828; Cell Signaling Technology), and rabbit anti-human LEF1 (catalogue 2230; Cell Signaling Technology). Co-IP was conducted as previously described [24 (link), 25 (link)]. Briefly, extracts from U87MG and U251 cells were immunoprecipitated with every 2 μg of mouse anti-flag tag antibody (catalogue F3165; Sigma‒Aldrich) for 4–6 h, followed by overnight incubation with protein A/G agarose beads (catalogue sc-2003; Santa Cruz Biotechnology). The conjunct complex was then washed, resuspended in protein sample buffer and subjected to SDS-PAGE. Then, target interacting proteins were revealed by immunoblot. Uncropped images for western blots are provided in the Supplementary Material.