Nitrocellulose membrane

Protein (20 µg/line) was separated on 10% gels, and the resulting gels were transferred to nitrocellulose membranes (Millipore, Molsheim, France). Primary antibodies diluted in TBST were used (overnight, 4 °C): rabbit polyclonal to CEP55 (#23891-1-AP, Proteintech, 1 to 2,000), rabbit monoclonal to SLC7A11 (#12691, Cell Signaling Technology, 1 to 1,000), rabbit monoclonal to ILF3 (#ab92355, Abcam, Cambridge, UK, 1 to 20,000), rabbit monoclonal to GPX4 (#ab125066, Abcam, 1 to 5,000), and rabbit polyclonal to β-actin (#ab8227, Abcam, 1 to 4,000). The anti-rabbit HRP secondary antibody (#ab6721, Abcam, 1 to 10,000) was used at room temperature for 1 h. Blot analysis was conducted using Clarity ECL solution (Bio-Rad, Marnes-la-Coquette, France) and Fluorochem M imaging system (Protein Simple, San Jose, CA, USA).

Whole cell lysates were extracted with RIPA lysis buffer (Fdbio Science) containing the protease inhibitor PMSF (Beyotime) followed by sonication. The supernatant was harvested by spinning at 13,300 rpm for 10 min at 4 °C. Protein concentrations were measured by bicinchoninic acid protein assay (Thermo Scientific). Equal amounts of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transferring onto nitrocellulose membranes (Millipore), blocking with 5% milk and incubating with antibody overnight at 4 °C, followed by incubation with fluorescence secondary antibodies, detecting by Odyssey (LI-COR).
For co-IP, cells were lysed in lysis buffer (Beyotime) containing PMSF (Beyotime) and cocktail (MCE), followed by vortexing for 30 min, with 15 s of oscillations every 5 min. After centrifugation samples were rotated incubating with Anti-FLAG M2 Magnetic Beads (Sigma) or Anti-HA Magnetic Beads (Sigma) overnight at 4 °C and washed beads by wash buffer (Beyotime) several times, boiling samples in loading buffer with denaturant SDS. Equal amounts of total protein were immunoprecipitated with antibodies. Precipitates were analyzed by immunoblotting. An aliquot of each lysate was used as input control.
For endogenous co-IP assay, add the antibody to the pre-prepared cell lysate at the recommended antibody-to-protein ratio, and incubate overnight at 4 °C to allow sufficient binding of the antibody to the target protein. A Protein A/G immunoassay (coprecipitation) kit (Biolinkedin) was used to incubate with the antigen-antibody mixture for 3 h at room temperature. The mixture was washed by PBST three times and denatured by boiling with denaturant SDS.

Western blotting was performed as previously described. Briefly, cells were lysed in an ice-cold radio immunoprecipitation assay lysis (RIPA) buffer (Solarbio, #R0010) containing protease inhibitors. The protein concentration of lysates was quantified using a bicinchoninic acid (BCA) protein assay kit from Solarbio (#XYW-3). Equal amounts of protein were loaded onto sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA) after separation. The membrane was blocked with 5% non-fat milk at room temperature for 1 h, followed by incubation with the primary antibody at 4 °C overnight. After incubation with the corresponding secondary antibody (MBL), the target protein was visualized by chemiluminescence. The primary antibodies and concentrations used for Western blotting were HSP70 (abmat, Shanghai, China, #M20033), DLAT (ABclonal, Wuhan, China, #A14530), ATP7B (proteintech, Shanghai, China, 19786-1-AP; abmart, Shanghai, China, T58616), CTR1 (abmart, T510261F), MTF1 (abmart, MG775483), beta-actin (ABclonal, #AC004), and GAPDH (ABclonal, #A19056).

Cultured cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Beyotime) supplemented with protease inhibitor (APExBIO, Houston, TX, USA) and phosphatase inhibitor (APExBIO). Protein quantification was performed using the Pierce™ BCA Protein Assay Kit (23227, ThermoFisher Scientific). Whole lysate samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the nitrocellulose membranes (Millipore, Burlington, MA, USA). After being blocked by 5% nonfat dried milk for 2 h, membranes were incubated with the specified primary antibodies overnight at 4 °C and combined with HRP-conjugated goat anti-rabbit or goat anti-mouse antibodies (Jackson ImmunoResearch Inc.) for another 2 h at room temperature. The images were then visualized using ChemiDoc+ (Bio-RAD, Hercules, CA, USA) after incubation with ECL (ShareBio, Shanghai, China). Signal intensity was detected and quantified by ImageJ software.

Briefly, cells (~1 × 10⁶) were lysed in RIPA buffer (150 mM NaCl, 25 mM Tris, pH 8.0, 1% Na-Deoxycholate, 0.5% SDS, 1% NP-40, 10mM Glycero-phosphate, 1mM Sodium fluoride, 2mM Sodium orthovanadate, 1mM EDTA, and Protease Inhibitors freshly added). An equal amount of protein was resolved using Novex 8–16% Tris-Glycine Gels (Invitrogen) and then transferred onto Nitrocellulose membrane (Millipore) followed with specific antibodies application. Antibody signal was detected using Immobilon Forte Western HRP Substrate (Millipore) and Luminescent Imager 680 (Amersham Bioscience). Primary antibodies were used: anti-HRP-b-Actin (Sigma, A23852), rat anti-LANA HHV8 (Abcam, ab4103), mouse Phospho-RNA Pol II Ser2 (Active Motif, 61083), mouse Phospho-RNA Pol II Ser5 (Invitrogen MA1–460093), rabbit Phospho-HisH3, Ser10 (Invitrogen, 701258).