Sds page gel

Sodium dodecyl‐polyacrylamide gel electrophoresis was performed with the cell‐free extract after sonication and each supernatant and resuspended pellet after ultracentrifugation following sonication (10 µL samples). The samples were incubated at 95 °C for 5 min after addition of SDS loading dye. After centrifugation, the samples were loaded onto an SDS‐PAGE Gel (GenScript, USA) with a protein ladder of 10 to 200 kDa (PageRuler, pre‐stained Thermofischer). The gels were run at 120 V on a Mini‐PROTEAN® Tetra Vertical Electrophoresis Cell (Bio‐Rad). After the run, the gel was rinsed with water and stained with Coomassie InstantBlue™. The overexpression levels were estimated to 10–20% for TmCHMO‐PTDH, 20–30% for AcCHMO‐PTDH and 10–20% for RhCHMO‐PTDH.

The cells or tissues were lysed in IP lysis buffer or RIPA lysis buffer (Beyotime, Beijing, China) containing a complete protease inhibitor cocktail (Roche, Basel, Switzerland). Whole-cell lysates were centrifuged with 12,000 × g for 10 min at 4 °C and the supernatant were used for immunoprecipitation via incubation with Atp6v0d1 antibody at 4 °C overnight, followed by incubation with Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, 88802, MA, USA) for 1 h to affiliate the antigen sample/antibody mixture. The magnetic beads were then washed three times with IP buffer and one time with purified water. After being eluted, immunoprecipitates were boiled in SDS loading buffer for 5 min at 95 °C and separated by SDS–PAGE Gel (Genscript, Nanjing, China), and transferred onto polyvinylidene fluoride membrane (Millipore, MA, USA). Western blot experiments were performed with the indicated antibodies and visualized by WesternBright^TM ECL kit (Advansta, CA, USA).

Samples were loaded on an 8% SDS-Page gel (Genscript) with 4×10⁶ infected RBCs or 50 µg protein for the parasite lysate approach. Proteins were transferred to a nitrocellulose membrane that was blocked with 5% skim milk (Sigma) in PBS overnight and incubated with rabbit antiserum against AcAS (1:1000) for 1 h. Subsequently, membranes were washed three times with PBS-Tween for 5 min, followed by incubation with secondary horseradish peroxide (HRP)-conjugated goat anti-rabbit antibodies (Dako P0448, 1:1000) for 1 h. Blots were then washed three times with PBS-Tween for 5 min and twice with PBS. Following a 5-min incubation with Clarity Max Western ECL Substrate (BioRad), protein blots were imaged using the ImageQuant LAS4000 (GE Healthcare). The band intensity was quantified using Fiji software.

The amount of CYP21A2 expressed was measured from the total protein extraction. Cells were incubated for 1 h with the lysis buffer previously described [46 (link)] and centrifuged at 15,000× g for 20 min and 4 °C. The supernatant was collected for the measurement of total protein through a Pierce Coomassie Plus (Bradford) Assay Kit (Thermo Fisher, Hanover Park, IL, USA). Seven µg of total protein were loaded on an SDS-PAGE gel (GenScript, Piscataway, NJ, USA) and then transferred to a PVDF membrane, as previously described [46 (link)]. Two primary antibodies were used at the same time: a mouse monoclonal DKY-Tag antibody diluted 1:1000 (GenScript, Cat# A00187) and a mouse monoclonal anti-β-Actin antibody diluted 1:1500 (Sigma Aldrich, St. Louis, MO, USA, Cat# SAB3500350). The secondary antibody, IRDye 800CW-conjugated donkey-anti-mouse (LI-COR, Nebraska, USA-Cat# 926-32212) was diluted at 1:15,000. An Odyssey SA Infrared Imaging system (LI-COR Bioscience Inc. Lincoln, NE, USA) was used to detect the fluorescence signal.

After the transfected HepG2 cells were washed with precol phosphate buffer saline (PBS), the cells were lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (Fdbio, Hangzhou, China) and an additional 1% protease inhibitor mixture (Fdbio, Hangzhou, China), and protein extraction was performed. We used the BCA Protein Detection Kit (Fdbio, Hangzhou, China) to detect protein concentrations. Proteins were separated on SDS-PAGE gel (Genscript, Nanjing, China), applied to PVDF film (Millipore, Burlington, VT, USA) and sealed with 5% skim milk powder. FOXO1 (ab179450, Abcam, UK) and GAPDH (ab8245, Abcam, Metropolis, UK) were used to incubate the product. After washing with TBS-Tween 20 buffer, the secondary antibody (Genscript, Nanjing, China) was incubated for 1 h at room temperature. Electro-chemi-luminescence (ECL) solution (Fdbio, Hangzhou, China) was developed using a FluorChem E chemiluminescence gel imaging system (ProteinSimple, Minneapolis, MN, USA). The final results were analyzed using ImageJ software.

BMDCs that were cultured with or without ADJ (1%) or LPS (100ng/ml) were washed with cold PBS and lysed in RIPA buffer (CST) containing protease and phosphatase inhibitor cocktail. Total protein levels in each lysate were estimated using Pierce BCA protein assay kit (Thermo Scientific; 23227). Samples containing 30-40ug protein were loaded and resolved on a 12% SDS-PAGE gel (Genscript), transferred to PVDF membrane using iBlot 2 Gel Transfer (Thermo Fisher Scientific; IB24001), blocked with 5% BSA in TBST for phospho-proteins and 5% milk for total proteins in TBST for 1 hour and probed with primary antibodies (at 4C overnight). Primary rabbit antibodies were used as follows at 1:1000 dilution in 5% BSA in TBST: rabbit monoclonal anti-phospho-Akt (Ser473; Clone: D9E), rabbit polyclonal anti-total Akt, rabbit monoclonal anti-phosphor-p70 S6 kinase (Thr389, Clone: 108D2), and rabbit polyclonal anti-p70 S6 kinase antibodies. Blots were extensively washed with TBST and incubated with goat anti-rabbit IgG (H+L)-HRP antibodies (Thermofisher) diluted in 5% non-fat milk in TBST for 1 hour at room temperature. Protein bands were visualized by ECL prime western blotting detection reagent (GE Healthcare.) Membranes were stripped with mild stripping buffer (0.1% SDS, 0.1% Tween 20, 1.5% Glycine), when necessary.