Anti pi3k

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.

AML-12 cells are preserved in the School of Pharmacy, Anhui Medical University. Anti-NUP85 was bought from Santa Cruz Biotechnology (CA, USA). Anti-CCR2 was bought from Abcam (Cambridge, UK). Anti-phosphorylated protein kinase B (p-AKT), anti-AKT, anti-phosphorylated phosphatidylinositol-3 kinase (p-PI3K) and anti-PI3K were bought from Immunoway (Suzhou, China). Anti-β-actin, anti-acyl-CoAoxidase-1 (ACOX-1) and anti-interleukin-1beta (IL-1β) were got from Proteintech (Wuhan, China). Anti-sterol regulatory element binding protein-1 (SREBP-1C) was purchased from Affinity Biotechnology (Liyang, China). Anti-peroxisome proliferator-activated receptor alpha (PPAR-α), anti-interleukin-6 (IL-6) and anti-tumor necrosis factor-alpha (TNF-α) were got from Wanleibio (Shengyang, China). IL-1β, IL-6 and TNF-α ELISA kits were purchased from ELK Biotechnology CO., Ltd. (Wuhan, China). Oil red O staining kit was purchased from Solabio (Beijing, China). LY294002 and ISRIB were bought from Med Chem Express (Shanghai, China). Alanine aminotransferase (ALT), TG and aspartate aminotransferase (AST) kits were bought from Mindray (Shenzhen, China).