Beta actin

Cells were washed with cold PBS, lysed with lysis buffer containing 25 mM tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, mixed with 5% SDS loading buffer, and heated for 5 minutes. The samples were electrophoresed with SDS PAGE and subsequently transferred to nitrocellulose membrane (GE healthcare). The membrane was blocked with 5% skim milk having Tween 20 (0.1%) and probed overnight with antibodies IFI6 (Abclonal, Cat No, A6157), HA-tag (abcam, Cat No. ab9110), beta-actin (abClonal, Cat No, AC028). The signals were detected with an enhanced chemiluminescence (ECL) substrate (Millipore, Billerica, MA).

After drug treatment of DMSO, Erastin, RSL3, staurosporine or combination with minocycline (Sigma, St. Louis, MO, USA), veliparib, rucaparib, liproxstatin-1, the cells were lysed with NETN buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA, plus 1 tablet of Roche complete protease inhibitor per 10 mL) to collect the total protein of the cells. After SDS-PAGE, the protein on the gel was transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA), sealed with 5% milk for 1 h, and the primary antibody GPX4 (ABclonal, 1:1000 dilution), HO-1 (Proteintech, Chicago, IL, USA, 1:1000), PAR (Trevigen, Gaithersburg, MD, USA, 1:1000), Flag (Sigma, 1:3000), GAPDH (ABclonal, 1:5000), beta-actin (ABclonal, Wuhan, Hubei, China, 1:5000), or cleaved caspase-3 (Cell Signaling Technology, Boston, MA, USA, 1:1000) were incubated overnight at 4 °C. The secondary antibody with HRP was incubated at room temperature for 1 h. Proteins were visualized with the SuperSignal Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

After a drug treatment using DMSO, Erastin, RSL3, Sorafenib, or in combination with liproxstatin-1, the cells (CAL33, CNE-2, S18, S26, AMC-HN-8, TU686) were lysed with NETN buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1mM EDTA, plus one tablet of Roche complete protease inhibitor per 10 mL) to collect the total protein of the cells. After SDS-PAGE, the protein on the gel was transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA) and sealed with 5% milk for 1 h. Then, the primary antibody GPX4 (ABclonal, 1:1000 dilution), HO-1 (Proteintech, Chicago, IL, USA, 1:1000), GAPDH (ABclonal, 1:5000), beta-actin (ABclonal, Wuhan, China, 1:5000), or cleaved caspase-3 (Cell Signaling Technology, Boston, MA, USA, 1:1000) were incubated overnight at 4 °C. The secondary antibody with HRP was incubated at room temperature for 1 h. Proteins were visualized with the SuperSignal Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).