Smurf2

Antibodies used included: SIRT2 (Abcam, Cat. No. ab211033), CTGF (Abcam, Cat. No. ab6992), FN1 (ABclonal, Wuhan, China; Cat. No. A12977), COL1A1 (Santa Cruz, Cat. No. sc-59772), TGF-β1 (Abcam, Cat. No. ab92486), β-actin (bioss, Beijing, China; Cat. No. bs-0061R), COL3A1 (Santa Cruz, Cat. No. sc-514601), E-cadherin (BD Biosciences, Cat. No. 610182), α-SMA (Santa Cruz, Cat. No. sc-84326), p-SMAD2 (Cell Signaling Technology, Cat. No. 18338), SMAD2 (Abcam, Cat. No. ab40855), acetyl Lysine (Abcam, Cat. No. ab190479), Ubiquitin (Novus, Cat. No. NB300-129), SMURF2 (ABclonal, Cat. No. A2278), CREBBP (ABclonal, Cat. No. A14237), P/CAF (ABclonal, Cat. No. A0066), Goat Anti-Rabbit IgG H&L (HRP) (Abcam, Cat. No. ab6721), and goat anti-mouse IgG H&L (HRP) (Abcam, Cat. No. ab205719).

The nuclear and cytoplasmic fractions of HCT116 and SW620 cells transfected with CRLM1-OE were prepared using the nuclear and cytoplasmic extraction kit (Thermo, Waltham, MA) according to manufacture's instructions. HCT116 and SW620 cells transfected with or without CRLM1-OE and/or si-hnRNPK were lysed with RIPA lysis buffer (R0010, Solarbio, China) supplemented with protease inhibitors (Roche). Protein concentrations were measured using a BCA kit (Pierce, Rockford, IL). Then, the total protein was divided using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA). For blocking the membrane, 5% skimmed milk was applied for 1 h, followed by primary antibodies against hnRNPK (Proteintech), BBC3 (Sigma-Aldrich), SMAD6 (ABclonal), BMP7 (Proteintech) and SMURF2 (ABclonal) overnight at 4 °C. Following the PVDF membrane's washing with TBST, it was incubated for 2 h with the corresponding secondary antibody. The proteins were identified using Pierce SuperSignal West Pico Chemiluminescent Substrate (Termo Fisher, Waltham, MA), following the manufacturer's instructions. Internal references were HistoneH3 and GAPDH antibody.