Oxphos human wb antibody cocktail

Cells (10 × 10⁶) were kept on ice for 20 min in 0.15 ml of a buffer containing 150 mM NaCl, 20 mM Tris, 5 mM EDTA-Tris, pH 7.4 with the addition of 1% (v/v) Triton X-100, 10% (v/v) glycerol and protease inhibitor cocktail (Merck). Cell extracts were cleared by centrifugation at 18,000 × g for 20 min, 4 °C. Sample buffer (NuPAGE™ LDS Sample buffer, Invitrogen) supplemented with 12.5% v/v β-mercaptoethanol) was added to supernatants, and samples were separated by polyacrylamide gel (NuPAGE™, 12% Bis-Tris, Invitrogen) electrophoresis and transferred to nitrocellulose membranes. Blocking was performed with a PBS solution containing 5% (w/v) non-fat dry milk (AppliChem, Darmstadt, Germany). Antibodies for OXPHOS (OXPHOS Human WB Antibody Cocktail), prohibitin, Citrate Synthase, CyPD, IF1, and for β, α, b and OSCP subunits were from Abcam (Cambridge, UK), for γ subunit was from Genetex (Alton Pkwy Irvine CA, USA), for TOM20 was from Santa Cruz Biotechnology (Dallas, TX, USA), and the one against GAPDH was from Cell Signaling (Danvers, MA, USA). Detailed information on the specific antibodies used can be found on the key resource table. Band pixels of each replicate are normalized on band pixels of their proper loading control (β, prohibitin or GAPDH). Mean pixel ratios ± SEM are then shown proportionally to the mean of CTR samples, expressed as 100% or 1.

U1 and U937 cells (seeding density: 10 × 10⁶ cells/well) were either left untreated for 48 h or treated with DON (12.5 µM for 48 h), prostratin (untreated for 24 h followed by 6 µM prostratin for 24 h), or DON + Prostratin (12.5 µM DON for 48 h and 6 µM prostratin for last 24 h). At 48 h, activation of HIV was measured by intracellular staining of HIV-1 core antigen-FITC, KC57 as mentioned earlier. Cells were harvested, washed in PBS, and centrifuged. Cell pellets were processed for mitochondrial extraction using Mitochondria Isolation Kit for Cultured cell (Thermo Scientific) using a reagent-based method as per manufacturers guidelines, followed by measuring OXPHOS protein levels using the total OXPHOS Human WB antibody cocktail (Abcam) with mitochondrial loading control VDAC using the antibody VDAC clone B-6 (Santa Cruz Biotechnology). Relative protein quantification was performed using ImageLab version 6.0.1 (Bio-Rad Laboratories Inc), results analyzed using Mann–Whitney U-test or unpaired t-test and visualized using GraphPad Prism 8.4.3 (significance p < 0.05). All laboratory experiments were performed in three independent replicates. Analysis was performed using unpaired t-test or Mann–Whitney U-test and visualized using GraphPad Prism 8.4.3 (significance p < 0.05). Uncropped and unedited blot images are included as Fig. S10.