Anti march1

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.