A21020

Radio-immunoprecipitation assay (RIPA) buffer and phenylmenthanesulfonyl fluoride (PMSF) protease inhibitor were used here to extract the CPM cellular proteins. The Western blot assays were carried out as previously reported [27 (link)]. The primary antibodies used in this study were as follows: IGFBP2 rabbit polyclonal antibody (11065-3-AP; proteintech, Chicago, IL, USA; 1:300), MyHC mouse monoclonal antibody (B103; DHSB, Lowa City, IA, USA; 1:500), and β-Tubulin monoclonal antibody (A01030; Abbkine, California, CA, USA; 1:5000). HRP conjugated goat anti-rabbit IgG (A21020; Abbkine, USA; 1:10000) and HRP conjugated goat anti-mouse IgG (A21010; Abbkine, USA; 1:10000) were used as secondary antibodies.

Cultured cells were lysed for 30 min in ice-cold RIPA lysis buffer (Servicebio, Wuhan, China) containing protease inhibitors (Servicebio, Wuhan, China). Protein concentration was measured by the Coomassie brilliant blue G-250 (BioFroxx; neoFroxx GmbH) staining method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for protein electrophoresis. The protein was transferred to a polyvinylidene difluoride membrane. The PVDF membrane was blocked in 5% BSA for 1h and then incubated with diluted primary antibodies for 12 h at 4 °C. After washing with TBST three times, the PDVF membrane was incubated in diluted HRP Goat Anti-Rabbit IgG (1:5000, A21020, abbkine) or HRP Goat Anti-mouse IgG (1:5000, A21010, abbkine) at room temperature for 1h. The proteins were detected by the ECL system (Bio-Rad) and analyzed by Image Lab (6.0.1). All primary antibodies were shown as follows: GAPDH (1:2500, rabbit, YM1235, Immunoway), CDC42 (1:200, mouse, sc-8401, SANTA CRUZ BIOTECHNOLOGY).

Xenograft tumor tissues in paraffin sections were dewaxed and rehydrated. Next, endogenous peroxidase and nonspecific binding sites were blocked with 10% bovine serum albumin (AR0009, Boster, Wuhan, China) for 1 h. Then, rabbit anti-RRM2 (1:50, Abclonal, Wuhan, China) antibody was applied to all sections at 4 °C for 14 h. The following day, binding was conducted with the corresponding peroxidase-conjugated secondary antibody (A21020, Abbkine, Wuhan, China) and incubated at room temperature for 30 min. Then, the diaminobenzidine solution was used to detect the targeted antigen.

The total proteins were extracted from cells using radioimmunoprecipitation (RIPA) cell lysis buffer (BB-3209, BestBio Co., Ltd., Shanghai, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrically transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with the sealing solution for 1 h, followed by incubation at 4 °C overnight with the addition of following primary antibodies: rabbit polyclonal antibody to CD133 (1: 1000, ab198981), CD13 (1: 500, ab154116), Nanog (1: 500, ab80892), SOX2 (1: 1000, ab97959), OCT-4 (1: 1000, ab19857), rabbit monoclonal antibodies to STAT3 (1: 1000, ab68153) and p-STAT3 (1: 2000, ab76315) (Abcam Inc., Cambridge, MA, USA), and rabbit polyclonal antibody to CADM1 (1: 1000, A1892, ABclonal Biotech Co., Ltd., Cambridge, MA, USA). On the following day, the membrane was incubated with HRP conjugated goat anti-rabbit immunoglobulin G (IgG) (1: 5000, A21020, Abbkine, USA) at 37 °C for 1 h and developed with ECL reagent. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference, the relative protein levels of target proteins were expressed as the ratio of gray value of target band to that of internal reference band. Each experiment was conducted 3 times.