Ab32130

Total protein content was extracted from the tissues and cells using Protein lysis buffer (C0481, Sigma, St Louis, MO). Following 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane. The membranes were subsequently incubated with the following antibodies to HK2 (dilution ratio of 1 : 1000, ab104836, Abcam), mTOR (dilution ratio of 1 : 2000, ab2732, Abcam), p-mTOR (dilution ratio of 1:1000, ab109268, Abcam), Beclin1 (dilution ratio of 1 : 500, ab114071, Abcam), Bax (dilution ratio of 1 : 1000, ab77566, Abcam), Bcl-2 (dilution ratio of 1 : 1000, ab692, Abcam), 4EBP1 (dilution ratio of 1 : 1000, ab32130, Abcam), p-4EBP1 (dilution ratio of 1:1000, ab47365, Abcam), LC3A/B (dilution ratio of 1 : 1000, ab128025, Abcam), MMP-9 (dilution ratio of 1 : 1000, ab73734, Abcam) and β-actin (dilution ratio of 1:500, Beijing Cwbiotech Co., Ltd., Beijing, China) overnight at 4°C. Afterwards, the horseradish peroxidase (HRP) -labeled goat anti-mouse or anti-rabbit IgG (SPA131 or SA27, dilution ratio of 1 : 500, Solarbio, Beijing, China) was incubated with the membrane at room temperature for 1.5 h. The relative expression of the proteins was measured as previously described 51 (link).

Cells were harvested after treatment and lysed with a lysis buffer (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) containing a protease inhibitor cocktail. The protein concentration was determined with a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions. Protein lysates were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel for separation by electro-phoresis and then transferred onto a poly-vinylidene fluoride membrane (Millipore, Boston, MA). The membrane was immersed in 5% skim milk at 4°C overnight to block non-specific binding sites. Thereafter, the membrane was incubated with primary antibodies against GPX4 (1:1000, Abcam, Cambridge, MA, USA, ab125066), phosphorylated mTOR (p-mTOR, 1:1000, Abcam, ab109268), total mTOR (t-mTOR, 1:10000, Abcam, ab134903), p-S6K1 (1:500, Abcam, ab131436), t-S6K1 (1:5000, Abcam, ab32529), p-4EBP1 (1:1000, Abcam, ab75767), t-4EBP1 (1:1000, Abcam, ab32130) and β-tubulin (1:5000, Abcam, ab7291) at room temperature for 2 h. After washing with Tris-buffered saline with Tween 20 (TBST), the membrane was probed with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Jackson ImmunoResearch) at room temperature for 1 h. After elution, the expression levels of the proteins were analyzed via chemiluminescence and quantified by using ImageJ Software.