Sc 100761

Western blots were performed using standard protocols. Cell lysates were prepared using RIPA buffer. Protein samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk. The membrane was then incubated with anti-E-Cadherin antibody (1:1000, bs-10009R, Bioss, Beijing, China), anti-Vimentin antibody (1:1000, bs-0756R, Bioss, Beijing, China), anti-TRAF6 antibody (1:1000, bs-1184R, Bioss, Beijing, China), anti-N-Cadherin antibody (1:1000, AF5237, Beyotime, Shanghai, China), anti-RGS2 antibody (1:1000, sc-100761, Santa Cruz), anti-Flag antibody (1:1000, M185-3, MBL, Beijing, China) or anti-αTubulin antibody (1:5000, AF5012, Beyotime, Shanghai, China) at 4 °C overnight, followed by incubation with HRP-anti-rabbit or HRP-anti-mouse secondary antibody at room temperature for 1 h. Signals were detected using an ECL Kit (P0018AS, Beyotime, Shanghai, China) according to the manufacturer’s protocol.

Immunohistochemistry staining was carried out by using the Histostain-Plus Kit (Kangwei Reagents, Beijing, China) according to the manufacturer’s instructions as described previously^{29 (link)}. Briefly, paraffin-embedded placental sections or tumor sections with a thickness of 4 μm were deparaffinized and rehydrated in xylene and subsequently a graded series of ethanol. After antigen retrieval in 10 mM sodium citrate and 10 mM citric acid, tissue sections were treated with 3% H₂O₂-methanol solution to quench endogenous peroxidase followed by sequential incubation with normal goat serum for 30 min at room temperature, with a control rabbit or mouse IgG or a primary antibody against aromatase (sc-374176, Santa Cruz, CA), RGS2 (sc-100761, Santa Cruz, CA) or Ki-67 (bs-23103R, Bioss, China) at 4 °C overnight, and with HRP-labeled secondary antibody (Life Technologies) for 30 min. Diaminobenzidine (DAB) solution was used for color development, and the sections were counterstained with hematoxylin.

Protein was extracted from lung tissue using RIPA lysis buffer (Beyotime Biotechnology, China). Samples were electrophoresed and subjected to Western blot analysis using a primary antibody against RGS2 (Santa Cruz Biotechnology, RGS2 BC-43, sc-100761) and β-actin. Anti-mouse secondary antibodies (ZSGB Biotechnology, China) were used to capture images with chemiluminescence and fluorescence systems (Syngene U.S.).