Ez link sulfo nhs ss biotin

Following 48 h of incubation, transiently transfected HEK293 cells, previously washed twice with cold 1X PBS, were treated with EZlinkTM Sulfo-NHS-SS-Biotin (Thermo Scientific, Waltham, MA, USA) 0.5 mg/mL in cold 1X PBS for 15 min at 4°C. Subsequently, the cells were washed twice with 200 mM Glycine in cold 1X PBS and twice with cold 1X PBS to inactivate and remove the excess biotin, respectively. The cells were then lysed with 1X lysis buffer [50 mM HEPES pH 7.4; 150 mM NaCl; 1.5 mM MgCl₂; 1 mM EGTA pH 8.0; 10% Glycerol; 1% Triton X-100; 1X Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany)] for 1 h at 4°C. Cell lysates were centrifuged at 16,000 g at 4°C for 15 min. Two milligrams of the supernatant were incubated with 50 μL Streptavidin Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 h at 4°C, and the remaining supernatant was kept as the input. The beads were subsequently washed five times with 1X lysis buffer before elution with 50 μL of 2X NuPAGE sample buffer (Invitrogen, Carlsbad, CA, USA) plus 100 mM DTT at 37°C for 30 min. These biotinylated fractions were analyzed as TRPM4 expressed at the cell surface. The input fractions, analyzed as total expression of TRPM4, were resuspended with 4X NuPAGE sample buffer plus 100 mM DTT to give a concentration of 1 mg/mL and incubated at 37°C for 30 min.

To assess the cellular level and S368 phosphorylation status of Cx43 on cell surfaces and in whole lysates, LN215-Cx43 cells on 100 mm culture plates were biotinylated using EZ-Link^TM Sulfo-NHS-SS-Biotin (ThermoFisher Scientific, Cat. No. 21331) according to the manufacturer’s instructions followed by lysis with PBS containing 1% Triton X-100, 1x protease inhibitor cocktail (Roche), and 1x phosphatase inhibitor cocktail (Pierce). Protein concentration was determined using a BCA assay. To collect surface proteins, 500 μg of protein was bound to 20 μL of NeutrAvidin^TM Agarose (Pierce) followed by elution with 2x Laemmli sample buffer. The surface samples and 20 μg of whole lysates were analyzed using immunoblotting as previously described (Lee et al., 2016 (link)). To confirm the knockout of GJA1, whole lysates were prepared without surface biotinylation. Anti-total Cx43 (BD Biosciences, 612400) and anti-S369-phospho-Cx43 (Cell Signaling, 3511), anti-Na⁺-K⁺-ATPase (Abcam, ab185065), and anti-actin (Santa Cruz, sc-1615) antibodies were used. The protein band densities were measured using ImageJ software.

Cultured cells were biotinylated by incubating them at 4 °C for 1 hour with 500 μl of PBS containing 0.5 mg/ml EZ-Link^TM Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific). Then they were scraped in lysis buffer containing cocktail inhibitors as described above. After incubation at 4 °C for 15 minutes, lysates were centrifuged at 15000 rpm for 15 minutes at 4 °C, and the supernatants were incubated in 40 μl of MagnaBind Streptavidin beads (Thermo Fisher Scientific) at 4 °C for 1 hour. Beads were boiled with 40 μl of Laemmli sample buffer, and then 10 μl of the supernatant was loaded on polyacrylamide gels to be processed for Western blot analysis. Experiments were performed independently in triplicate.

Cells were starved with serum-free DMEM overnight, washed with ice-cold PBS, and incubated with PBS containing 0.5 mg/mL non-permeable EZ-link^TM Sulfo-NHS-SS-Biotin (pH 8.0; Thermo Scientific) on ice for 30 min. (EZ-link Sulfo-NHS-SS-Biotin is a membrane-impermeable reagent which forms a stable covalent linkage with an extended spacer arm to reduce the steric hindrance associated with avidin binding.) The liquid was suctioned. Cells were incubated with 4 mL of 100 mmol/L glycine at 4°C for 5 min. The step described above was repeated. Cells were washed and harvested by scraping in pre-cooled cell lysate buffer containing 1% Triton-X100 and protease inhibitors (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5). The supernatant was harvested and boiled in 4× buffer solution for 10 min. For pull down of biotinylated proteins, 500 μL of the protein extract was incubated with 30 μL of high-capacity streptavidin agarose beads (Neutravid Agarose; 29202; Pierce, Rockford, IL, United States) overnight at 4°C. Beads were washed, eluted by incubation in Laemmli buffer containing dithiothreitol at 100°C for 10 min, and analyzed by western immunoblotting. The lysate input and elutes were subjected to SDS–PAGE and analyzed by western immunoblotting.

To study the expression at the plasma membrane, biotinylation assay of membrane proteins was used. In this assay, membrane proteins are labeled with biotin and subsequently immunoprecipitated with streptavidin beads to isolate the protein membrane fraction. Mouse hippocampal HT22 cells (Merck Millipore, Burlington, MA, USA), previously incubated with 1 mM glutamate (Sigma Aldrich, Darmstadt, Germany), were treated with EZlinkTM Sulfo-NHS-SS-Biotin (Thermo Scientific, Waltham, MA, USA) 0.5 mg/mL in cold 1X PBS for 15 min at 4 °C. Subsequently, the cells were washed twice with 200 mM Glycine in cold 1X PBS and twice with cold 1X PBS to inactivate and remove the excess of biotin, respectively. The cells were then lysed and centrifuged at 16′000 g at 4 °C for 15 min. Two milligrams of the supernatant were incubated with 50 μL Streptavidin Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 h at 4 °C, while forty μg of protein were kept for the input fraction. The beads were subsequently washed five times with 1X lysis buffer before elution with 50 μL of 2X NuPAGE sample buffer (Invitrogen, Carlsbad, CA, USA) plus 100 mM DTT at 37 °C for 30 min.