Peroxidase affinipure goat anti rabbit igg h l antibody

To extract protein, ahRPE were washed and then lysed with RIPA buffer containing phosphatase and protease inhibitor cocktails (Roche) on ice. Samples were scraped on ice, agitated for 30 min at 4 °C and then centrifuged at 12,000 rpm for 20 min at 4 °C to remove debris. The supernatant was collected, aliquoted to prevent freeze-thaw cycles, and protein concentration was subsequently determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts were separated by Bolt^TM 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific) and transferred onto iBlot® 2 PVDF Mini Stacks (Thermo Fisher Scientific) membranes and were probed with antibodies (Supplementary Table 4). Proteins of interest were detected with Peroxidase AffiniPure Goat Anti-Mouse IgG, F(ab’)₂ Fragment Specific antibody (1:10000, Jackson ImmunoResearch, Cat #:115–035–072) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) antibody (1:10000, Jackson ImmunoResearch, Cat #:111–035–045), and subsequently visualized with ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) according to the provided protocol. All samples were stored at −80 °C. Uncropped western blots for Figs. 3d, 5c, d, and 6d can be found in Supplementary Figs 5 and 6.

Proteins in VLP containing samples were separated by SDS-PAGE using 4–12% Bis–Tris Nu-PAGE gels (Invitrogen) and electro-transferred to a PVDF membrane using the Trans-Blot Turbo Transfer Pack (BioRad). Membranes were blocked (1xPBS pH 7.4, 0.05% Tween20, 5% non-fat skim milk) and subsequently incubated with a rabbit anti-HIV-1 p55 + p24 + p17 antibody (Abcam, 1:2000) overnight at 4 °C. After washing, the membranes were incubated with Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) antibody (Jackson ImmunoResearch, 1:10,000) for 1 h at room temperature (RT), washed and developed using the SuperSignal West Pico PLUS Chemiluminescence Substrate (Thermo Scientific), and images were obtained using a Chemidoc^TMMP Imaging System (BioRad).

Following antibodies were used in this study: anti-p-Smad (CST, 13820S), anti-Smad (ProteinTech, 10429-1-AP), anti-p-STAT3 (ser727; CST, 9134S), anti-STAT3 (CST, 9139S), anti-SOCS3 (CST, 2923S), anti-myc (CST, 2276S), anti-β-actin (Santa Cruz, sc-1616), Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L) Antibody (Jackson ImmunoResearch Labs, 115-035-003), Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) Antibody (Jackson ImmunoResearch Labs, 111-035-003). For CO-IP experiment, the following agents and antibodies were used, Protein G PLUS-Agarose Antibody (Santa Cruz, sc-2002), Normal Mouse IgG Antibody (Santa Cruz, sc-2025), anti-myc (CST, 2276S), Peroxidase-AffiniPure Goat Anti-Mouse IgG, Light Chain Specific Antibody (Jackson ImmunoResearch Labs, 115-035-174).