Anti slc7a11

Cells were collected and lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), protease inhibitors). Equal amounts of proteins (40 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis gels and then transferred to a nitrocellulose blotting membrane (PALL Corporation, Mexico). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 °C with the respective primary antibody. The following antibodies were used: anti-c-MYC (1:1000, 13987S), anti-SLC3A2 (1:1000, 13180S), anti-E-cadherin (1:1000, 3195S), and anti-SLC7A11 (1:500, 12691S) from Cell Signaling Technology (Danvers, MA); anti-β-actin (1:10,000, sc-47778), anti-MTDH (1:250, 517220), and anti-GPx4 (1:500, sc-166570) from Santa Cruz Biotechnology; anti-vimentin (1:1000,v6389) from Sigma-Aldrich; anti-ZEB1 (1:1000, NBP1-88845) from Novus Biologicals USA. Membranes were further incubated with secondary antibody (1:10,000, 7074S or 7076S, Cell Signaling Technology) at room temperature for 2 h. Signal bands were detected using the Bio-Rad ChemiDoc system and densitometry were analyzed with Bio-Rad Image Lab Software (Bio-Rad Laboratories).

In our study, the cell samples were lysed using radioimmunoprecipitation assay lysis buffer (RIPA, Thermo Fisher Scientific), and the total protein concentration of different groups was detected using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). In our study, the cell lysates (20 μg/lane) were separated using 10% SDS-PAGE gel and then transferred to nitrocellulose membranes. The membrane was blocked with 5% nonfat dried milk diluted in PBS, and further incubated with primary antibodies overnight at 4 °C. Herein, the different primary antibodies used were: anti-SLC7A11 (1:3000; Cell signaling, Cat #: 12691), anti-GPX4 (1:1000; Santa Crus, Cat #: sc-166,570), anti-FTH (1:2000; Abcam, Cat #: ab65080) and anti-GAPDH (1:3000; Santa Cruz, Cat #: sc-47,724). The secondary antibodies used were: Anti-mouse IgG (HRP-conjugated; 1:5000; Sigma-Aldrich, Cat #: A-9044) and anti-rabbit IgG (HRP-conjugated; 1:5000; Sigma-Aldrich, Cat #: A-0545). Finally, the protein bands in each lane were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and ChemiDoc Imagers (Bio-Rad Laboratories). The results were finally quantified using the ImageJ 1.x software (National Institutes of Health). All of the raw, uncropped blots for images throughout the paper are shown in Supplementary Fig. 1.

Colon tissues were lysed by using the Cell Lytic Buffer supplemented with both proteases (PIC, Merck) and phosphatases (NaF 10 mM; Na₃VO₄ 1 mM) inhibitors. Equal amounts of protein lysates (20 mg) were subjected to SDS-Page separation and proteins electroblotted onto Nitrocellulose (Merck) membranes. Non-fat 5% dry milk (Merck) in PBS was used as a blocking solution, and indicated primary antibodies, in blocking solution were incubated o.n. at 4 °C. Appropriate HRP-conjugated secondary antibodies were diluted in blocking solution (1:5000) and incubated for 1 h at r.t. A Westar ANTARES ECL kit (Cyanagen, Bologna, Italy) was then used and the signal acquired by a ChemiDoc^TM Touch (Bio-Rad), and analyzed using Image Lab software (5.0; Bio-Rad). Primary antibodies were as follows: anti-Bip/Grp78 (1:500; Santa Cruz, Dallas, TX, USA); anti-Calnexin (1:500; Santa Cruz); anti-Calreticulin (1:1000; Abcam, Cambridge, UK); anti-PARP cleaved (1:500; Cell Signaling, Danvers, MA, USA); anti-SLC7A11 (1:250; Cell Signaling); anti-Occludin (1:500; Cell Signaling); anti-Caspase-3 (1:250; Santa Cruz); anti-aActin (1:5000; Sigma, Darmstadt, Germany); anti-bTubulin (1:5000; Santa Cruz). HRP-conjugated secondary antibodies were diluted in blocking solution (1:5000; Jackson ImmunoResearch, Cambridgeshire, UK).