Protein extraction and western blot analysis
dry milk
LYSED, SECONDARY, LYSIS
The cells were lysed using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, China). The protein concentration was determined by BCA protein concentration kit (Beyotime, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done in advance to separate the proteins. The proteins were subsequently electroblotted onto polyvinylidene difluoride membranes (Millipore, 150 Billerica, MA, USA). The membranes were blocked for 2 h in a solution of 5% nonfat dry milk in Tris Buffer Solution Tween (TBST). The primary antibodies ZIC2 (ZIC Family Member 2; HUABIO, 1: 2000) and β-actin (#YT0099, 1:5000) were then incubated at 4 °C overnight. The membranes were washed with TBST every 10 min. Membranes were washed and stained for 1 h at room temperature with goat anti-rabbit IgG secondary antibody (HUABIO, 1: 50,000). After that, the membranes were washed three times with TBST every 10 min. The blots were cut prior to hybridisation with antibodies during blotting. The bands were developed using an improved ECL kit (Thermo Scientific, Rockford, USA). GelDoc XR equipment was used to take the images, which were then analyzed using Image Lab Software (Bio-Rad). The original, uncropped Western Blot images of the study were in Additional File 3.