Ab38449

The proteins of SCMECs in each group were also extracted using radioimmunoprecipitation assay (RIPA) cold lysis buffer. Specific operational procedures and primary antibodies were in accordance with that described above. The primary antibodies include β-actin (1 : 1000; 3700S; CST), anti-PTEN antibody (1 : 1000; ab267787; Abcam), phospho-pan-AKT1/2/3 (Ser473) antibody (1 : 500; ab38449; Affinity), and anti-ZO1 tight junction protein antibody (1 : 1000; ab96587; Abcam).

First, we added 600 µL RIPA lysis buffer (P0013D, Beyotime, Shanghai, China) with phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor into cells. The total protein concentration of samples was determined using the BCA protein assay kit (pc0020, Solarbio, Beijing, China). The sample (20 µg) was separated by 10% SDS-PAGE and transferred to PVDF membrane (10600023, GE Healthcare Life, USA). After the membrane was washed with TBST 3 times, we blocked the PVDF membrane in 5% skim milk for 1.5–2 h. Then, we incubated the membrane with the primary antibody (anti-AKT: Abcam, ab8805, 1 : 500; anti-p-AKT: Abcam, ab38449, 1 : 1000; mTOR antibody: Affinity, AF6308, 1 : 1000; anti-S6K1: Abcam, ab32359, 1 : 1000; anti-LC3: Abcam, ab51520, 1 : 3000; and anti-beta actin: Abcam, ab8227, 1 : 4000) and secondary antibody (anti-rabbit IgG, HRP-linked antibody: CST, 7074, 1 : 3000; anti-mouse IgG, HRP-linked antibody: CST, 7076, 1 : 3000), separately. At last, the PVDF membrane was incubated with ECL luminescence reagent and analyzed with Chemi capture software.