P9416

Cells (1×10⁷) under 80% confluence or IVD tissues (0.1 g) were lysed in 1 mL ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher; #89901) containing the protease inhibitor (Abcam; #ab142778). Equal amounts (30 µg) of proteins were loaded into the wells of SDS-PAGE gel and separated by electrophoresis. Proteins were transferred onto the PVDF (polyvinylidene fluoride) membrane (Sigma-Aldrich; #IPSN07852) and blocked with 5% fat-free milk for one hour at room temperature. The membranes were then incubated with primary antibodies, including anti-BMPR1a (Abcam; #ab264043), anti-BMPR1b (Abcam; #ab175385), anti-BMPR2 (Abcam; #ab96826), anti-Smad1/5/8 (Sigma-Aldrich; #SAB2702532), anti-pSmad1/5/8 (Sigma-Aldrich; #AB3848-I), anti-Smad4 (Sigma-Aldrich; #HPA019154), anti-Puma (Abcam; #ab9645), anti-Apaf-1 (Abcam; #ab233786), anti-CASP9 (Abcam; #ab184786), anti-CASP3 (Thermo Fisher; #MA1-16843), anti-HDAC1 (Abcam; #ab7028), and anti-β-actin (Sigma-Aldrich; #A2066). After incubation at 4 °C overnight, membranes were washed 5 times with a PBS buffer containing 0.1% Tween-20 (Sigma-Aldrich; #P9416) and then probed with secondary antibodies (Abcam; #ab6721 and #ab6728). Protein signals were recorded by the Bio-Rad Gel Imaging System (Bio-Rad, Shanghai, China; #1708265).

Preparations of cell lysates for expression analyses essentially followed the guidelines in reference 28 (link). Cultures were pelleted by centrifugation and washed with HBSS-G (S. salmonicida) or PBS (G. intestinalis). Cells were normalized to 0.05 optical density (OD) units/10 µl at 600 nm in a spectrophotometer. Samples were boiled for 10 min in 1× Laemmli buffer with 100 mM dithiothreitol (DTT). Protein separations was performed using precast polyacrylamide gels (Bio-Rad Mini-Protean Any-kD TGX stain free, catalog no. 456-8125) and transferred to polyvinylidene difluoride (PVDF) (Bio-Rad catalog no. 162-0177). Membranes were blocked with 5% dry milk (Semper) in 0.05% Tween 20 (Sigma-Aldrich catalog no. P9416) in Tris-buffered saline (TBS) for 1 h. Proteins were detected with a primary mouse anti-V5 monoclonal antibody, SV5-Pk1 (Abcam catalog no. AB27671), and diluted 1:2,000 incubated at RT for 2 h and a secondary HRP-conjugated rabbit anti-mouse polyclonal antibody (Dako catalog no. P0161) diluted 1:10,000 and incubated at RT for 1 h. Developing was done using Clarity Western ECL substrate (Bio-Rad catalog no. 170-5061), and blots were imaged using the Bio-Rad ChemiDoc MP+ system. Images were processed using Bio-Rad Image Lab version 4.0 and Adobe Illustrator CC.