Rabmab

Staged embryos were fixed in 4% PFA in PBS, cryoprotected with 30% sucrose in PBS, and frozen in OCT on dry ice. Ten-micrometre cryosections were blocked using the mouse on mouse blocking reagent from Vector (cat. # MKB-2213), and then stained with antibodies for OSR1 (mouse monoclonal Santa Cruz cat. # 376545 at 1:40) and myogenin (Abcam RabMab cat. # ab124800 at 1:40). Secondary detection was done with InVitrogen donkey anti-rabbit Alexa 594 cat. # A21207, and InVitrogen goat anti-mouse Alexa 488 cat. # A11029, both at 1:300 dilutions. Sections were first screened on a Zeiss Axio Observer Z.1 and then imaged for deconvolution microscopy using a Leica DMI6000, with a 63× oil immersion lens, and Huygens Professional deconvolution software from SVI.

The cells were starved in an FBS-free medium for 24 h and then cultured in the absence or presence of arecoline for 48 h. The proteins were harvested using lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime Bio, Wuhan, China). Protein concentrations were examined and proteins were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated with the antibodies to TPM1 (RabMAb, 1:2000, Catalog Number ab133292; Abcam, Cambridge, MA, USA), Smad2/3 (1:750, Catalog Number WL0152; Wanleibio, Shenyang, China), and p-Smad2/3 (1:500, Catalog Number WL02305; Wanleibio). Alpha-tubulin antibody (1:5000, Catalog NumberRM2007; Rayantibody, Beijing, China) was used as an internal control. Chemiluminescent signals were visualised using ECL reagents (Cwbiotech, Beijing, China).

The total protein of each sample was harvested using RIPA lysing solution (Thermo Scientific, Rockford, IL, USA), which contained Protease Inhibitor Cocktail (Thermo Scientific). A BCA protein assay kit (Thermo Scientific) was utilized to determine protein concentration, and proteins were isolated with SDS PAGE and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Subsequently, Tris buffer, which contained 0.1% Tween-20 and 5% skimmed milk, was used to block the membrane at 4°C overnight. Antibodies, including anti-TPM1 (RabMAb1:5000, Abcam, Catalog Number ab109505, Cambridge, MA, USA) and anti-GAPDH (1:5000, Bioworld, Catalog Number AP0060, Minneapolis, MN, USA), were used to incubate the membranes and then respective secondary antibodies (horseradish peroxidase-conjugated, 1:50000, Bioworld, Catalog Number BS13278, Minneapolis, MN, USA), conjugated with horseradish peroxidase, were used to bind the first antibodies. The results were obtained using an ECL detection system (Thermo Scientific), according to the instructions of the manufacturer.