Plasma membrane protein isolation kit

Plasma membrane fractions of 293T cells transfected with different Envs were isolated using the Plasma Membrane Protein Isolation kit (Abcam) following the manufacturer’s protocol. PM fractions were resuspended in lysis buffer (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Triton-X, 1 mM DTT and protease inhibitors) and immunoprecipitated (with rotation) O/N at 4 °C with bNAbs. Next day the mixture was incubated (with rotation) for 1 h at 4 °C with Immobilized Protein G resins (G Biosciences) and washed three times with PBS + 1% Triton-X. Washed beads were analyzed by western blot analysis using HIVIG Ab.
CD4-induced shedding of gp120 was assayed as described previously [23 (link)]. Briefly, cells transfected with plasmids expressing JRCSF Env were harvested and washed with FACS buffer 2 and then incubated with or without 50 µg/ml sCD4-183 (NIH AIDS Reagent) for 1 h at 4 °C with intermittent mixing. Cells were centrifuged and the supernatant subjected to ELISA as described previously [23 (link)] except that mAb, 39F was used as the primary antibody (1:1000 dilution) and peroxidase-coupled goat anti-human IgG (Jackson ImmunoResearch, Cat No. 109-036-008) was used as secondary antibody.

The membrane protein extractions from the aortic media (VSMCs were the only cell type in this layer) or cultured VSMCs were carried out by the Plasma Membrane Protein Isolation Kit (Abcam, UK) according to the manufacturer's instructions. Total proteins were also extracted, and western blot was performed as described in our previous reports [12 (link), 13 (link)]. The expressions of NOX subunits and mitogen-activated protein kinase (MAPK) signaling pathway were detected using their specific antibodies: anti-NOX1 (1 : 2000, ab55831, Abcam, Cambridge, UK), anti-NOX2 (1 : 500, ab80508, Abcam, Cambridge, UK), anti-p47phox (1 : 1000, ab795, Abcam, Cambridge, UK), anti-NADPH oxidase organizer 1 (anti-NOXO1, 1 : 500, sc-390927, Santa Cruz Biotechnology Co., Ltd., Japan), anti-Rac1 (1 : 1000, sc-95, Santa Cruz Biotechnology Co.), anti-p38 (1 : 1000, ab31828, Abcam), anti-phospho-p38 (p-p38, 1 : 1000, ab47363, Abcam), anti-ERK1/2 (1 : 1000, ab17942, Abcam), anti-phospho-ERK1/2 (p-ERK1/2, 1 : 1000, 9101, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-JNK (1 : 1000, ab179461, Abcam), and anti-phospho-JNK (p-JNK, 1 : 1000, ab124956, Abcam). To ensure equal protein loading, β-actin (1 : 5000, ab8226; Abcam) and Na⁺/K⁺-ATPase (1 : 100000, ab76020, Abcam) were used as loading controls for the cytoplasm and plasma membrane, respectively.

Membrane protein extraction from C2C12 skeletal muscle cells was carried out by Plasma Membrane Protein Isolation Kit (Abcam, UK) according to the manufacturer’s instructions. Protein extraction and western blot were performed as previously described (19 (link)). Briefly, total proteins were extracted from liver and muscle (gastrocnemius muscle) tissues or C2C12 skeletal muscle cells using a RIPA lysis buffer (Beyotime Biotech Inc., China) supplemented with complete EDTA-free protease inhibitor cocktail tablets (Roche) according to the manufacturer’s instructions. The protein concentrations were determined using the BCA Protein Assay Kit (Applygen, China). Proteins (40 μg) were fractionated by gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore), blocked with 5% nonfat milk, and then incubated with primary antibodies overnight at 4°C. After 3 washes with phosphate-buffered saline plus 0.1% Tween-20, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Signals were tested by ECL reagent and protein expressions were calculated by normalization to β-actin.