Gapdh

Cell lysates were prepared in PN lysis buffer (10 mM PIPES, 50 mM NaCl, 150 mM sucrose, 50 mM NaF, 40 mM Na₄P₂O₇10H₂O, 1 mM Na₃VO₄, 1% Triton X-100, Complete protease inhibitors (Sigma)). Total protein (30 µg) was separated by SDS-PAGE, and transferred to nitrocellulose using the Trans-Blot Turbo Transfer System (Bio-Rad). Blots were blocked in blocking buffer 5% bovine serum albumin (BSA, Fisher) in TBS-T (TBS-T is 50 mM Tris-Cl, 150 mM NaCl, pH 8 with 0.1% Tween20) for 1 hour at 22 ^oC, then incubated overnight at 4 ^oC with primary antibodies diluted in blocking buffer. Blots were further incubated with secondary goat anti-mouse or -rabbit fluorophore-conjugated antibodies (Dylight 800 or Dylight 680, ThermoFisher) in 2% non-fat milk in TBS-T, and scanned on the Licor Odyssey.
The following primary antibodies were used: pY705-STAT3, pS727-STAT3, and STAT3 (Cell Signaling Technologies); and GAPDH (Biolegend). In some experiments, cells were treated with IL-6 at the indicated concentrations and time prior to preparation of cell lysates.

Cells were lysed in PN buffer (PN is 10 mM PIPES, 150 mM sucrose, 50 mM NaCl, 50 mM NaF, 40 mM Na₄P₂O_7.10H₂O, 1 mM Na₃VO₄, 1% Triton X-100, and complete protease inhibitors (Sigma, Oakville, ON, Canada)). Total protein (30 µg) was separated by SDS-PAGE, transferred to nitrocellulose, and blots blocked in 5% bovine serum albumin (BSA, ThermoFisher) in TBS-T (TBS-T is 50 mM TrisHCl pH 8, 150 mM NaCl, 0.1% Tween 20) for 1 h at 22 °C, then incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. Blots were then incubated with fluorophore-conjugated goat anti-mouse or -rabbit antibodies (Dylight 680 or 800, ThermoFisher) in 2% non-fat milk in TBS-T, and imaged on the Licor Odyssey (Lincoln, NE, USA).
The following primary antibodies were used: pY705-STAT3, STAT3, pY1034/1035-JAK1, JAK1, pY1008-JAK2, JAK2, pY1054/1055TYK2, TYK2 (Cell Signaling Technologies, Danvers, MA, USA); and GAPDH (Biolegend, San Diego, CA, USA). In some experiments, cells were treated with IL-6, LIF, OSM, or IL-11 (Genscript) at the indicated concentrations and time prior to the preparation of cell lysates.

To determine cellular and viral protein expression, infected purified CD4⁺ T cells or PBMCs were washed in phosphate-buffered saline (PBS), lysed in Western blot lysis buffer (150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 0.1% NP-40, 500 mM Na₃VO₄, 500 mM NaF [pH 7.5]), and cleared by centrifugation at 20,800 × g for 20 min at 4°C. Lysates were mixed with protein sample loading buffer supplemented with 10% β-mercaptoethanol and heated at 95°C for 5 min. Proteins were separated on NuPAGE 4% to 12% Bis-Tris gels, blotted onto Immobilon-FL polyvinylidene difluoride (PVDF) membranes, and stained using primary antibodies (Abs) directed against HA (catalog number ab18181; Abcam), V5 (catalog number ab206571; Abcam), HIV-1 p24 (catalog number ab9071; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 607902; BioLegend); Vpu subtype C antiserum (catalog number 11942; NIH Reagent Program); polyclonal rabbit anti-HIV-1 Vpu Ab 32-81 (kindly provided by Ulrich Schubert); and infrared dye-labeled secondary antibodies (IRDye; Li-Cor). A signal enhancer Hikari kit for Western blotting and enzyme-linked immunosorbent assays (ELISAs) (Nacalai Tesque) was used for Vpu and p24 staining. Proteins were detected using a Li-Cor Odyssey scanner, and band intensities were quantified using Li-Cor Image Studio Lite version 3.1.

Whole-cell lysates were separated on NuPAGE 4–12% Bis-Tris Gels (Invitrogen) for 90 min at 120 V and blotted at constant 30 V for 30 min onto 0.45-µm Immobilon-FL PVDF membrane (Merck Millipore). After the transfer, the membrane was blocked in 1% casein in PBS (Thermo Scientific) and stained using primary antibodies directed against strep (1:5000, Thermo Fisher Scientific, PA5-119611), V5 tag (1:1000, Cell Signalling, #13202), VSV-M (1:2000, Absolute Antibody, 23H12, #Ab01404-2.0), or GAPDH (1:1000, BioLegend, #631401). Infrared Dye labeled secondary antibodies IRDye 800CW Goat anti-Mouse #926-32210, IRDye 800CW Goat anti-Rat (#926-32219), IRDye 680CW Goat anti-Rabbit (#925-68071), IRDye 680CW Goat anti-Mouse (#926-68070), IRDye 800CW Goat anti-Rabbit (#926-32211) were used, all 1:10,000. Proteins were detected using a LI-COR Odyssey scanner. Uncropped blots are shown in Supplementary Fig. 9.

Antibodies were purchased from the following sources: phospho-NF-κB, NF-κB, phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, and COX-2 from Cell Signaling Technology (Danvers, MA, USA), HO-1 from Abcam (Cambridge, UK), NRF2 and TLR4 from Cusabio (Wuhan, China), iNOS from Invitrogen (Carlsbad, CA, USA), MyD88 from Novus Biologicals (Centennial, CO, USA), and GAPDH from BioLegend (San Diego, CA, USA).