Ab227518

Total protein was extracted by RIPA buffer, and protein concentration was detected using a BCA protein quantification kit. A 30 μg protein sample was used for 10% SDS-PAGE electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane, and the membrane was blocked with 5% BSA. Primary antibody was added to the membrane and incubated overnight, and then incubated with an HRP-labeled secondary antibody, and ECL development was performed after the incubation. The antibodies were as follows: COL1A1 (Abcam, #ab34710)^{9 (link)}, RUNX2 (Abcam, #ab192256)^{10 (link)}, OCN (Abcam, #ab13418)^{11 (link)}, p-SMAD1 (Abcam, #ab73211)^{12 (link)}, t-SMAD1 (Abcam, #ab33902)^{13 (link)}, CDH11 (Abcam, #ab151302)^{14 (link)}, VEGF (Abcam, #ab52917)¹⁵ , beta-catenin (Abcam, #ab32572)^{16 (link)}, Dicer (Abcam, #ab227518), and GAPDH (Abcam, #ab181602)^{17 (link)}.

Total protein was extracted by RIPA, and protein concentration was detected by a bicinchoninic acid protein quantification kit^{11 (link),12 (link)}. A 30 μg protein sample was used for 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane, and the PVDF membrane was blocked with 5% bovine serum albumin. Then, primary antibody was added and incubated overnight, after which an incubation with an HRP-labeled secondary antibody and ECL development were performed. The following primary antibodies were used in this study: COL1A1 (Abcam, #ab34710), RUNX2 (Abcam, #ab192256), OCN (Abcam, #ab13418) ZEB1 (Abcam, #ab245283), VEGF (Abcam, #ab52917), beta-catenin (Abcam, #ab32572), Dicer (Abcam, #ab227518), and GAPDH (Abcam, #ab181602).

Cells were lysed on ice in RIPA protein lysate solution for 30 minutes. The cell debris was pelleted by centrifugation for 10 min at 10,000 rpm. Protein concentrations in the supernatant were determined using the bicinchoninic acid (BCA) method. Samples in sample buffer were heated for 10 min at 98° C. Samples containing 20 μg protein were resolved by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels using a vertical transfer system (#1658033 Bio-Rad Laboratories Inc.). The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Nonspecific protein binding on the membranes was blocked with 5% BSA for 1 h and the membranes were incubated overnight at 4° C with the primary antibodies. The membranes were then washed with TBST for 1 h, incubated with HRP-labeled secondary antibodies, and finally washed with TBST. Bound proteins on the blots were visualized using an automatic chemiluminescence imaging analysis system (#5200; Tanon Science and Technology Co., Ltd., Shanghai, China). The primary antibodies specific to COL1A1 (#ab34710), RUNX2 (#ab192256), OCN (#ab13418), p-SMAD1 (#ab73211), t-SMAD1 (#ab33902), RUNX2 (#ab151302), VEGF (#ab52917), beta-catenin (#ab32572), Dicer (#ab227518), and GAPDH (#ab181602) were purchased from Abcam (Cambridge, UK).