Ecl reagents

After culture for 4 days, cells were lysed in RIPA lysis buffer (Solarbio, Beijing, China) and 1 mM PMSF (Solarbio, Beijing, China) on ice. The concentration of protein was metered using the BCA Protein Assay Kit (Thermo Scientific, MA, USA) and heat denaturation of protein in protein sample loading buffer (EpiZyme, Shanghai, China) at 100 °C for 10 min. Equal quantities of protein were separated on 10% SDS-PAGE gels by electrophoresis, transferred onto 0.45 um polyvinylidene-fluoride membranes (PVDF) (Immobilon, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in TBST buffer pH 7.5 for 1 h at room temperature, incubated with the first antibody. Anti-GAPDH, PI3K p85, PI3K p110, AKT, phosphorylated-PI3K p85 and phosphorylated-AKT were used as primary antibodies. All antibodies are listed in Additional file 6: Table S1. Membranes were washed with TBST 3 times, 10 min each time, and incubated with corresponding secondary antibodies at room temperature for 1 h. Protein bands were visualized by ECL reagents (TransGen, Beijing, China).

Animal samples were prepared from different developmental stages, different temperatures, and salt concentrations. Total proteins were extracted from each sample using RIPA lysis buffer and quantified by the BCA protein assay kit. Protein samples (80 μg each) were subjected to fractionation by SDS–PAGE and transferred to Polyvinylidenefluoride (PVDF) membranes. The PVDF membrane was blocked with 5% skim milk powder for two hours at room temperature and allowed to incubate with the original antibody overnight at 4 °C. Rabbit anti–As–RAD9 polyclonal antibody and GAPDH antibody were diluted 1:500 and 1:1000 with PBST, respectively. The membrane was washed three times with PBST and then incubated with HRP-conjugated secondary antibody for one hour at 37 °C and then washed three times with PBST and once with PBS. Reactive protein bands on the membrane were visualized using ECL reagents (Transgen, Beijing, China) in a dark room. Image grayscale analysis in Image J software was used to compare the density (corresponding to intensity) of the bands on the Western blot, and the resulting data were used to construct a histogram. The intensity of expression of a particular protein band is normalized to the GAPDH band. Other antibodies, such as RAD17, RAD1, CHK1, and HUS1 were purchased from BOSTER (Wuhan, China) according to sequence homology, with all homology exceeding 80%.