Nupage 4 12 bis tris gradient gel

HEK293T cells were harvested 48 h post transfection and PBMCs were harvested 72 h post transduction by lysing them in Western Blot lysis buffer (150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 0.1% (v/v) NP-40, 0.5 mM Na₃VO₄, 0.5 mM NaF, pH 7.5). After addition of Protein Loading Dye (LI-COR) and 2.5% β-mercaptoethanol, samples were incubated at 95 °C for 5 min, separated on a NuPAGE Bis-tris 4–12% gradient gel (Invitrogen) and blotted onto an Immobilon-FL PVDF membrane (Merck Millipore). The membrane was blocked in 5% milk and proteins were stained using primary antibodies directed against HIV-1 Nef (NIH #2949; dilution 1:200), GAPDH (BioLegend #631401; dilution 1:200) and HIV-1 p24 (abcam #ab9071) and secondary antibodies conjugated with Infrared Dyes (LI-COR). Bands were detected using the infrared imager Odyssey9120 (LI-COR) and the Image Studio Lite Version 4.0 (LI COR).

Samples were loaded on a NuPAGE Bis-Tris 4%–12% gradient gel (Invitrogen) and the Coomassie stained bands were pooled and in gel digested with trypsin and GluC, respectively, as described elsewhere [62 (link)]. LC-MS analyses of the peptides were done on an EasyLC nano-HPLC (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) as described elsewhere [63 (link)]. MS data were processed using the software suite MaxQuant, version 1.2.2.9 [64 (link)] and searched using Andromeda search engine [65 (link)] against a target-decoy E. coli database containing 4,311 forward protein sequences, the sequences of the tagged and overexpressed proteins and 248 frequently observed protein contaminants. Trypsin or GluC, were set as proteases in which two missed cleavage sites were allowed. Carbamidomethylation of cysteine was set as fixed modification; N-terminal acetylation, methionine oxidation and serine/threonine/tyrosine phosphorylation were set as variable modifications. Initial precursor mass tolerance was set to 6 parts per million (ppm) at the precursor ion and 20 ppm at the fragment ion level. False discovery rates were set to 1% at peptide, phosphorylation site, and protein group level.

Protein fractionation was also carried out using polyacrylamide gel electrophoresis. Proteins were loaded and separated on a NuPage Bis-Tris 4–12% gradient gel (Invitrogen) based on the manufacturer’s instructions. The gel was stained with Coomassie Blue and cut into 16 slices. Resulting gel slices were destained by three washes with 10 mM ammonium bicarbonate (ABC) and ACN (1:1, v/v) followed by reduction with 10 mM DTT in 20 mM ABC for 45 min at 56°C. Subsequent alkylation was carried out with 55 mM IAA in 20 mM ABC for 30 min at RT in the dark followed by two washes with 5 mM ABC and once with 100% ACN. The gel slices were next dehydrated in a vacuum centrifuge. Proteins were digested with Lys-C (Wako; 12.5 ng μL^-1 in 20 mM ABC) at 25°C overnight. Resulting peptides were extracted in a three step procedure: Step 1- with 3% TFA in 30% ACN; step 2- with 0.5% acetic acid in 80% ACN; step 3- with 100% ACN. Samples were evaporated in a vacuum centrifuge and peptides were desalted using stage-tips (as described before) and subjected to nano-LC-MS/MS measurements on the LTQ-Orbitrap Elite (Thermo Fisher Scientific).

Protein separation via SDS-PAGE followed by an In-Gel digestion was used for one biological replicate from each Super-SILAC experiment. Briefly, 100 μg (50 μg SSS and 50 μg light) of crude protein extracts were separated on a NuPage Bis-Tris 4–12% gradient gel (Invitrogen). After staining the gel with Coomassie Blue, protein lanes with cut into 12 equal slices. Coomassie Blue stain was subsequently removed from each gel slice via washing with 10 mM ammonium bicarbonate (ABC) and acetonitrile (ACN) (1:1, v/v). Protein slices were reduced with 10 mM dithiothreitol (DTT) in 20 mM (ABC) for 45 min at 56°C and alkylated with 55 mM iodoacetamide IAA in 20 mM ABC for 30 min in the dark at room temperature. Gel slices were washed twice with 5 mM ABC, followed by a dehydration step by incubating with 100% ACN at room temperature. Proteins were digested with Lys-C (Wako) (12.5 ng/μL in 20 mM ABC) at 37°C overnight. Digested peptides were recovered from each gel slice using three consecutive extraction steps: (I) 3% TFA in 30% ACN (II) 0.5% acetic acid in 80% ACN (III) 100% ACN. Resulting peptides were desalted using StageTips (Ishihama et al., 2006 (link)).

Cells were grown in 10 cm dishes and on the day of experiments incubated with 0.5 mM DSS or PBS for 30 min in RT. Cross-linking was quenched by addition of 1M Tris, cells washed with 3 times with 1X PBS and lysed in RIPA buffer. For cell surface biotinylation 10-15x10⁶ cells were washed twice with ice cold PBS and re-suspended in 1 ml of 1X PBS pH 8. Sulfo-NHS-SS-Biotin was added to a final concentration of 0.4 mM and cells were incubated in RT for 30 mins following three washes with 1X PBS and cell lysis in RIPA buffer.
Equal amount of total cells lysate (500 μg) was applied to 40 ul of GFP-nanobody conjugated beads or strepavidin magnetic beads (NEB) and incubated on a rotating shaker for 1 hr in 4 °C. Beads were then washed four times with 500 ul of RIPA buffer and 40 ul of 1X SDS loading buffer, containing reducing agents was added to the beads, heated to 100 °C for 10 min and resolved on NuPAGE Bis-Tris 4−12% gradient gel (Invitrogen). Gels were stained with Coomassie for Proteomics analysis (see Proteomics analysis) or transferred to PVDF membranes and processed for immunoblotting as previously described [47 (link)].