Sds page sample loading buffer

RIPA Lysis Buffer (Beyotime) containing phosphatase and proteinase inhibitors (Beyotime) was used to lysed cells washing by cold PBS three times, and then BCA protein assay kit (Beyotime) was utilized to determine the protein concentration. Cell lysates were mixed with SDS‐PAGE sample loading buffer (Epizyme), followed by boiling for 5 min at 100°C. After that, protein (40 µg/lane) was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membranes. After blocked membranes with 5% skim milk for two hours at room temperature, primary rabbit antibodies against NEIL1(ab192517, 1:1000) and GPADH (AF1186, 1:1000) were incubated on a shaker overnight at 4°C. Following Tris‐buffered saline Tween‐20 (TBST, Epizyme) washing, the membranes were then incubated with secondary antibodies against rabbit for two hours at room temperature. At last, protein bands were examined by ECL solution (Thermo Scientific) and the bands density was analyzed by ImageJ software.

We added liquid nitrogen to 20–50 mg tissue, grinded it into powder, and then added an appropriate amount of protein lysis buffer (Beyotime). Whole‐cell protein lysates were extracted using protein lysis buffer, and the protein concentrations of tissue and cell were determined by the BCA assay (Solarbio). The lysates were boiled in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) sample loading buffer (EpiZyme) for 5–10 min at 99°C and separated on SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Millipore) by electroblotting, and after blocking in 5% nonfat milk (Sangon Biotech) for 2–3 h. Then the membrane was incubated with primary antibodies against anti‐MARCH1 (Immunoway), anti‐AKT (ImmunoWay), anti‐PI3K (Immunoway), anti‐pAKT (Immunoway), anti‐pPI3K (Immunoway), anti‐E‐cadherin (Cell Signaling Technology), anti‐Vimentin (Proteintech), anti‐β‐actin (Proteintech), anti‐tubulin‐α (Cell Signaling Technology) at 4°C overnight and then left with the secondary antibodies peroxidase‐conjugated goat anti‐rabbit (BOSTER) or peroxidase‐conjugated goat anti‐mouse (BOSTER) for 30 min at 37°C. Finally, the membranes were quantified using an enhanced chemiluminescence signal (ECL, EpiZyme). Photometric analyses of immunoblots were carried out using the Image Lab software package. The quantitative analysis through ImageJ software.