Ez link sulfo nhs ss biotin

After washing mature adipocytes five times with cold PBS (Life Technologies, Carlsbad, CA, USA) containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS-CM), the cells were labeled twice with 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce Chemical Co., Rockford, IL) on ice for 20 min. Non-reacted biotin was quenched and removed by washing with Tris-buffered saline-CM for 5 min. The cells were lysed with RIPA buffer on ice for 30 min and centrifuged at 4 °C and 12,000×g for 30 min. After quantification, the supernatants of the cell lysates were incubated with NeutrAvidin Agarose Resin overnight at 4 °C and pelleted by centrifugation. The pellets were collected with SDS loading buffer and analyzed using western blotting, as described above.

Surface proteins were labeled with EZ-Link SULFO-NHS-SS-biotin (1 mg/ml, Pierce Chemical Co., Rockford, IL, USA) for 1 h as described before^{21 (link)}. Cells were then rinsed with PBS containing 100 mM glycine thoroughly to quench unreacted biotin and then lysed in modified radio-immuno-precipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8; 150 mM NaCl; 1% Triton X-100 and 1% sodium deoxycholate; 10 μg/ml leupeptin; 100 μg/ml TPCK; and 1 mM PMSF). Proteins (150–300 μg) were incubated overnight at 4 °C with end-over-end shaking in the presence of Streptavidin beads (Pierce Chemical Co.). Beads were thoroughly washed, resuspended in 30 μl loading buffer, and analyzed with Western blots.

cDC1, cDC2, and CD14⁺ DCs that have been exposed to different stimuli were washed to remove the remaining virus, poly I:C, or gardiquimod. Then they were 1: 5 mixed with allogeneic PBMCs that were labeled with CellTrace Violet. After 5 days of incubation, cells were harvested and stained for CD3, CD4, and CD8a.
For determining the frequency and proliferation of Tregs, only DCs exposed to viral suspensions were examined. In this case, half of the supernatant of the original DC culture was kept with cells for mixing with PBMCs. After 5 days, cells were collected and incubated with an anti-CD25 antibody (Bio-Rad, Spain) biotinylated by EZ-Link Sulfo-NHS-SS-Biotin (Fisher Scientific, Spain); then, streptavidin PerCP-Cy5.5 was added followed by a CD4a-FITC antibody. Foxp3 intracellular staining was performed with an anti-Foxp3 PE antibody using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, United States). Proliferation was determined by examining the intensity of CellTrace Violet fluorescence.

Cell surface proteins were biotinylated as described [24] (link). Briefly, neurons were washed twice with ice-cold PBS and cell surface proteins were biotinylated by incubation with 1 mg/ml of EZ-Link Sulfo-NHS-SS-Biotin (Fisher, Pittsburgh, PA) for 30 min at 4°C. Neurons were washed 3 times and lysed in RIPA Buffer (0.15 mM NaCl/0.05 mM Tris-HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS). The cell lysates were centrifuged at 10,000×g for 15 min at 4°C. Biotinylated surface proteins were isolated by immune-precipitating with Streptavidin magnetic beads (Fisher) for 1 h at 4°C, rinsed three times and dissociated in SDS sample buffer. The eluted samples were collected and analyzed by immunoblotting. Some of the cell lysates were analyzed for total GluA1 and GluA2/3 levels, and the ratios of surface/total GluA1 or GluA2/3 levels were calculated.

UNC5B-FL-HA, UNC5B-Δ8-HA, and L1CAM-HA overexpressing HeLa cells were incubated with EZ-Link Sulfo-NHS-SS-Biotin (Life Technologies, #21331) 0.2 mg/ml in chilled CM-PBS at 4 °C for 30 min. After three washes (10 min each) in quenching buffer (100 mM glycine, 2.5 mM CaCl₂, 1 mM Mg₂Cl₂ in PBS) cells were collected by scraping and lysed in RIPA buffer (1% Triton X- 100, 1% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 59 mM NaF, 160 mM NaCl, and 20 mM Tris-HCl, pH 7.4) supplemented with protease inhibitors (PI) (cOmplete™ and EDTA-free Protease Inhibitor cocktail; Roche). Biotinylated proteins were retrieved by using Pierce High Capacity Streptavidin agarose beads (Life Technologies, #20359). After three washed in RIPA + PI buffer, biotinylated proteins were eluted in a 2× SDS-sample buffer by heating at 95 °C for 10 min. Eluted proteins were loaded in an SDS-PAGE gel and analyzed by immunoblotting with the indicated antibodies. A small fraction of protein lysate for each condition was saved as an input fraction.

Biotinylation of surface protein was performed as explained before. Briefly, thioglycollate-elicited peritoneal macrophages from WT and Angptl4^−/− mice were plated on six-well plates and incubated with or without Ac-LDL (120 μg ml⁻¹) for 24 h. Cells were then washed with PBS⁺⁺ (1 × PBS supplemented with 0.02 mM CaCl₂ and 0.15 mM MgCl₂) and incubated for 30 min on ice with 250 μM EZ-link SulfoNHS-SS Biotin diluted in PBS++ (Life Technologies). Cells were again washed with PBS⁺⁺ and the reaction was quenched for 30 min on ice in quenching buffer (PBS++ supplemented with 100 mM glycine). Cells were scraped and 1/5^th of suspension were set aside as whole lysate and rest of the biotin-modified proteins were immunoprecipitated with NeutrAvidin agarose beads (Life Technologies) overnight at 4 °C. Biotin-modified proteins were collected by centrifugation at 5,000g for 5 min. Intracellular, unmodified proteins were collected from the supernatant of the 5,000g spin. The streptavidin beads were washed three times in PBS⁺⁺ before proteins were removed from the beads by incubation at 42 °C for 20 min, in 2 × SDS sample loading buffer supplemented with β-mercaptoethanol. Biotinylated cell surface ABCA1 was detected by immunoblotting total surface proteins against ABCA1 antibody (1:1000; Abcam; clone AB.H10; # ab18180).

To determine the surface fraction of NBCn1, MDCK-II cells were cultured on Transwell filters for 4 days in order to allow polarization. Cells were incubated for 30 min at 4 °C with freshly made 0.2 mg/ml cell impermeable EZ-Link Sulfo-NHS-SS-Biotin (Life Technologies, #21331) diluted in PBS added to either the basolateral or apical side of the filters as indicated. Removal of excess biotin was done by several washing steps in cold quenching buffer (0.1 M glycine in PBS). Cells were lysed in cold RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 0.5% Sodium deoxycholate, 1% Igepal CA630 and Complete^TM mini protease inhibitor). Samples were centrifuged for 15 min at 16.000 RCF and 4 °C, and the supernatant was adjusted to equal amounts of protein (DC assay, BioRad). A small fraction of the supernatant was dissolved in 4 × LDS sample buffer and saved as the Total Lysate Fraction (TLF). The remaining supernatant was incubated with prewashed streptavidine-conjugated agarose beads (Sigma-Aldrich, #S1638) for 2 h with gently rolling at 4 °C. Samples were washed several times in cold RIPA buffer, dissolved in 2 × LDS sample buffer and heated for 5 min at 95 °C. Beads were pelleted by centrifugation at 4 °C for 4 min at 2000 RCF and the supernatant was saved as the Pull-down (PD) fraction. Samples were processed for Western blotting as below.