Hrp labeled secondary antibody

The liquefied semen was centrifuged to discard the seminal plasma, and the pellets were resuspended with 1 × SDS-PAGE loading buffer including β-mercaptoethanol in the ratio of 10⁶ sperm to 100 μl loading buffer before they were boiled for 5 min. The supernatant proteins were stored at −80 °C with aliquots for the western blotting. In each sample, 10 μl proteins were separated by 12.5% SDS-PAGE and semi-dry blotted to PVDF (Polyvinylidene Fluoride) membranes (Millipore, Bedford, MA, USA). Blocked 2 h within 1 × NET, the membranes were incubated with primary rabbit polyclonal antibody to DEFB126 (1:1000; Santa Cruz, California, USA) overnight at 4 °C and then incubated with the HRP-labeled secondary antibody (1:10000; Cwbiotech, Beijing, China) for 1 h at RT. The bands, when washed thrice in TBST, were detected by ECL kit (GE Amersham, Pittsburgh, USA). The semi-quantification of target protein and reference protein were analyzed by ImageJ 1.48 gray scale scanning software.

Ishikawa cells which had received the different treatments were scraped from the six-well plates and lysed with RIPA lysing buffer (Beyotime; Nantong, China) containing 1 mM PMSF and a protease inhibitor cocktail (Beyotime). The lysed cells from each treatment group were centrifuged at 16,000g for 20 min, and the supernatant fractions were collected. Next, a 20 μg sample of supernatant protein was separated by 8 % SDS-PAGE, and then semi-dry blotted onto PVDF (polyvinylidene fluoride) membranes (Millipore, Bedford, MA, USA). After being blocked for 2 h with TBST containing 5 % non-fat dry milk, the membranes were incubated overnight at 4 °C with a primary rabbit monoclonal antibody to integrin β1 (1:5000) or integrin β3 (1:5000); after which, they were incubated with the HRP-labeled secondary antibody (1:10,000; Cwbiotech, Beijing, China) for 1 h at room temperature. HRP-labeled β-actin (1:10,000; Sigma-Aldrich) was used as an internal control protein. The protein blots were developed using Beyo ECL Plus reagent (ThermoFisher Scientific; Waltham, MA, USA), and the results were recorded with a gel imaging system. The relative expression levels of integrins β1 and β3 in the different samples were calculated using a Gel-Pro-Analyzer (Media Cybernetics; Rockville, MD, USA).

For detection of protein levels, Western blot analysis was performed as previously described (Yin et al., 2018 (link)). Cultural cells were washed three times by cold PBS and then digested by cell lysis buffer (Beyotime, P0013, Haimen, China) with 1% protease inhibitor cocktail (Calbiochem, 539134, Darmstadt, Germany) on ice for 30mins. Protein concentrations were analysed by BCA protein assay kit (Thermo Fisher Scientific, 23225). An equal amount of protein for each sample was subjected to SDS-PAGE using 5% stacking gel and 12% separating gel, 120V, 1h and transferred (400mA, 2h) to nitrocellulose filter membranes (Pall, 66485, Port Washington, NY). Membranes were blocked with 5% skimmed milk (BD Biosciences, 232100, Franklin Lakes, NJ) for 1 hour at room temperature, and then incubated with primary antibodies at 4°C overnight with the following primary antibodies: SMAD2 (Rabbit pAb, 1:1000; Proteintech, 12570-1-AP, Hubei, China), Ser465/Ser467 phosphorylated SMAD2 (Rabbit pAb, 1:700; Bioss, bs-3419R, Beijing, China) and GAPDH (Rabbit pAb, 1:1000; Proteintech, 10494-1-AP). Blots were then incubated with HRP-labeled secondary antibody (1:2000; CWBIO, CW0103, Beijing, China) and visualized using chemiluminescence detection system (Thermo Fisher Scientific, NCI5080). Protein bands were exposed to X-ray film (Kodak, 6535876, Rochester, NY). GAPDH was adopted as internal control.