Hrp conjugated anti rabbit or anti mouse igg

Cell lines were lysed with RIPA cell lysate, the total protein was extracted, and then the BSA protein quantification kit (CWBIO, China) was used to determine the protein concentration of the sample. Protien samples were separated using 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred from the gel onto a PVDF membrane (Millipore, Bedford, MA). Skimmed milk powder was diluted with TBST at a 3%concentration, blocking the membrane for 1 hour. The primary antibodies Mouse Monclonal-GAPDH (TA-08, ZSBio, Beijing, China, dilution 1:2000) and Rabbit Anti Myc (ab32072, Abcam, 1:1000) was added and incubated overnight, the secondary antibody(HRP-conjugated anti-rabbit or anti-mouse IgG at the appropriate dilutions Beyotime, Nanjing, China) was incubated for 2 hours and the PVDF membranes were washed with TBST solution. After adding the luminescent solution, the PVDF membrane was detected using the Ultra-High sensitivity chemiluminescence imaging system (BIORAD, Shanghai, China)

The tissue samples of eggshell gland, kidney, duodenum, jejunum, and ileum were put into 1 mL of RIPA buffer (Beyotime, China), and homogenized in ice bath. Then, it was centrifuged at 4°C, 12000 g for 10 min. The protein concentration was detected by a BCA protein assay kit (Beyotime, China). The protein samples were separated by 10% SDS–PAGE (Bio-Rad, Richmond, 246 CA) and then transferred to PVDF membranes (Millipore, Boston, MA), at 200 mA and 2 h at 4°C. After blocking with western blocking buffer (Beyotime, China) for 1 h at room temperature, the membranes were incubated with anti-Calbindin-d28k (Sigma, Boston, MA), anti-PMCA (Thermo, Waltham, MA), anti-β-actin and anti-β-tubulin (Beyotime, Jiangsu, China) overnight at 4°C. The membranes were then washed in 1 × TBST buffer and probed with the secondary antibody (HRP-conjugated anti-rabbit or anti-mouse IgG, Beyotime) diluted at 1:1000, at 4°C for 4 h. The bands were then visualized with Hyperfilm ECL reagent (Beyotime, Jiangsu, China) using BioSpectrum 810 (UVP LLC, Jena, Germany) (Uerlings et al., 2018 (link); Wang et al., 2020 ).