Ab140010

Total proteins were lysed using RIPA lysis buffer (Sigma-Aldrich) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Abcam, Cambridge, United Kingdom), the non-specific protein binding was blocked by immersing the membranes in 5% skim milk (Thermo Fisher Scientific) for 3 h. Then the membranes were incubated with anti-B cell lymphoma-2 (anti-Bcl-2; Abcam, ab32124, 1:1000), anti-Bcl-2-Associated X (anti-Bax; Abcam, ab32503, 1:1000), anti-Cleaved-Caspase3 (anti-C-Caspase3; Abcam, ab32503, 1:1000), anti-GREM1 (Abcam, ab140010, 1:1000) and anti-GAPDH (Abcam, ab9485, 1:3000) at 4 °C overnight. PVDF membranes were washed using 0.05% PBS with Tween 20 (PBST; Invitrogen), followed by the incubation of the secondary antibody (Abcam, ab205718, 1:5000) at room temperature for 45 min. Ultimately, the protein bands were detected using the enhanced chemiluminescence reagent (Sigma-Aldrich) and the protein expression was analyzed under the Image J software (NIH, Bethesda, MD, USA).

The total protein of cells to be detected was extracted using Radio Immunoprecipitation Assay (RIPA) cell lysis buffer (BB‐3209; BestBio Co., Ltd., Shanghai, China), followed by electrophoretic separation with sodium dodecyl sulphate polyacrylamide gel electropheresis (SDS‐PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with solution for 1 hour and added with primary antibody of rabbit anti‐human clone antibody for incubation at 4°C overnight: GREM1 (1:1000, ab140010), TGF‐β1 (1:200, ab92486), Smad4 (1:1000, ab40759), p21 (1:1000, ab109520), E‐cadherin (1:10 000, ab40772), Vimentin (1:1000, ab92547), Snail (1:500, ab53519) and GAPDH (1:10 000, ab181602) (all the antibodies were purchased from Abcam, Cambridge, UK), followed by incubation with gentle shaking for 1 hour at 37°C along with IgG goat anti‐rabbit secondary antibody. Then, the membrane was developed, and the relative expression of target proteins calculated as the ratio of the grey value of the target protein band to the grey value of internal control band in the same sample, where GAPDH served as internal control. The relative expression of proteins was calculated as the staining intensity of the target protein/the corresponding GAPDH staining intensity. Each experiment was conducted in triplicate, with the average value calculated.