Azure c300 gel imaging system

Cells were lysed in RIPA Buffer (Sigma) containing the Phosphatase Inhibitor Cocktail and Protease Inhibitor Cocktail (Roche), and kept on ice for 40 min vortexing every 5 min. Afterward, samples were centrifuged at 13,000 rpm for 25 min at 4 °C. Cell lysates were quantized with Protein Assay Dye Reagent Concentrate (Bio-Rad) and separated by SDS-PAGE with Bolt Bis-Tris Plus gels (4–12% polyacrylamide concentrations) in MES SDS Running Buffer (Invitrogen). PageRuler™ Prestained Protein Ladder (Thermo Scientific™) was used as size standards of proteins. The proteins were transferred on nitrocellulose membrane using the iBlot 2 Dry Blotting System with iBlot 2 Transfer Stacks (Invitrogen). Membranes were blocked for 1 h with 5% Milk (Skim Milk Powder, Millipore) in TBS-Tween 0,1% (TBS, Bio-Rad; Tween 20, Sigma-Aldrich) and then incubated overnight with anti-GPX4 (Cell Signaling, #52455) or anti-β-actin (Sigma, A5441) primary antibodies. The next day, membranes were incubated with secondary antibody HRP-conjugated and developed with ECL West Pico Plus Substrate (Immunological Sciences). Images were acquired with Azure c300 Gel Imaging System (Azure Biosystems). All results were normalized over β-actin.

Newman wild-type and Δspa MVs of equal amounts (2 µg total protein) and LPS standard from E. coli serotype 055:B5 (250 µg/mL) were mixed with 0.5 × volumes of Laemmli sample buffer (65.8 mM Tris–HCL, pH 6.8, 26.3% w/v glycerol, 2.1% SDS, and 0.01% bromophenol blue; Bio-Rad) and boiled for 5 min prior to loading on 4–20% SDS-PAGE gradient gels (Mini-PROTEAN TGX precast gels; Bio-Rad). Precision Plus Protein Unstained Protein Standards (Bio-Rad) were used as molecular weight markers. To stain LPS, the gels were stained using the Pro-Q Emerald 300 LPS gel staining kit according to the manufacturer’s instructions (Thermo Fischer Scientific). The presence of glycoproteins was examined by staining the gels with SYPRO Ruby protein gel stain according to the manufacturer’s instructions (Thermo Fisher Scientific). Stained LPS and protein bands were visualized under UV light using an Azure c300 gel imaging system (Azure Biosystems).

Cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 5 mM EDTA, 30 mM Na₂HPO₄, 50 mM NaF, and 1 mM Na₃OV₄), and protein concentrations were determined using the Pierce^TM BCA Protein Assay Kit (cat# 23225, Thermo Fisher Scientific). Samples containing equal amounts of cell lysate were loaded onto SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blotted with a corresponding primary antibody in blocking buffer (Tris-buffered saline/Tween-20 containing 5% skim milk) and incubated with horseradish peroxidase-conjugated secondary antibodies. The signal of the band was detected by the Azure C300 gel imaging system (Azure Biosystems, Dublin, CA, USA).

Samples of lung adenocarcinoma participants were gathered for protein detection. RIPA buffer with protease inhibitor cocktail (Roche, Basel, Switzerland) was used for sample preparation and the supernatants were then resuspended in sample buffer. After denaturation of the samples by the way of boiling for 10 min, 30% polyacrylamide gel was used for protein electrophoresis with the same amount (40 μg). Whereafter, PVDF membrane was used for protein transfer (Millipore, Massachusetts, USA). Blocking buffer was used to block membranes for 2 h, then the following antibodies were used SEPT9 (Abcam [Cambridge, USA] ab38314, 1:500), HIST1H2BH (Abcam ab1790, 1:1000); MAPT (Abcam ab64193, 1:500), GAPDH (Goodhere [Hangzhou, China] AB‐P‐R 001, 1:1000), and finally overnight under the condition of 4°C. The membranes were in a state of HRP‐linked secondary antibodies (Boster [Wuhan, China] BA1054, 1:50,000) for 2 h after cleaned by TBST for three times on the following day. An electrochemiluminescence (ECL) detection kit (Thermo [Waltham, USA] NCI5079) was used for immunoreactive protein bands observation at the completion of washing. Azure c300 Gel Imaging System (Azure Biosystems, Dublin, USA) and BandScan software was used to scan and quantify protein bands. Three times have been done for all of the experiments.

Nuclear extracts were isolated from HEK-293T cells (RRID: CVCL_0063) with the nuclear extraction kit (Solarbio, Beijing, China). Electrophoretic mobility shift assay (EMSA) was performed with Chemiluminescent kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The probe including biotin-labeled, unlabeled, and mutant ones for C/EBPα-specific binding were synthesized by General Biol (Hefei, China), and the sequences are listed in Table S5. After being annealed to double-stranded oligonucleotides, the probes were incubated with 20 µg nuclear extracts for 20 min at room temperature. In the competition group, unlabeled oligonucleotides were first incubated for 10 min before the addition of labelled probe. The mixture was electrophoresed on 6.5% polyacrylamide gel at 90 V for 1.5 h, and then transferred to a positively charged nylon membrane (Beyotime). The gels were visualized on Azure c300 Gel Imaging System (Azure Biosystems, Dublin, CA, USA).