Nupage 4 to 12 bis tris gel

Bacterial cultures were grown overnight in 10 mM Mg²⁺, washed 3 times in medium lacking Mg²⁺, and subcultured 1:50 in 10 ml of low or high Mg²⁺ medium for the indicated times. Insoluble protein isolation was performed as described [81 (link)]. Total, soluble, and insoluble protein samples were electrophoresed on NuPAGE 4% to 12% Bis-Tris gels (Thermo Fisher Scientific) and stained with Coomassie blue. Quantification of protein amounts was performed using the gel analysis feature of ImageJ to measure intensities and abundances of protein bands present in each lane.

Protein samples (∼50-μg proteins/lane) were resolved on NuPAGE 4 to 12% Bis-Tris gels (Thermo Fisher Scientific). Prestained Protein Standards (Bio-Rad) were used as molecular weight markers. Membrane raft fractions were analyzed by immunoblotting using chemiluminescence as described previously (33 (link)). Other Western blot analyses were performed using the indicated primary antibodies, followed by Infrared IRDye-labeled secondary antibodies, and visualized using the Odyssey Infrared imaging system (LI-COR Biosciences). In most cases, blots were cut between molecular weight markers to allow simultaneous immunostaining and reprobing of the top and bottom parts with distinct antibody species. Band intensity was determined using the associated Image Studio Lite, version 5.0, Software (LI-COR Biosciences) to accurately quantify protein expression levels. Anti-actin antibodies were used to normalize for protein loading. Anti-pTyr⁴¹⁶ SFK antibodies were used to assess the phosphorylation state of Fyn, which was determined after normalizing the pSFK signal corresponding to Fyn (determined as being the fastest migrating band) for total Fyn expression levels and protein loading. PP2A demethylation was assessed as described previously (33 (link)).

For Western blot, 2 μg of purified complexes was run on NuPAGE 4 to 12% bis-tris gels (Thermo Fisher Scientific), transferred to Immobilon-P membranes (Millipore), and probed with anti-Strep (ab180957, Abcam, 1:5000) and anti-HA (3F10, Roche, 1:5000) antibodies in 5% milk–phosphate-buffered saline (PBS)/0.01% Tween overnight at 4°C. Membranes were then washed and incubated with horseradish peroxidase anti-rabbit (Santa Cruz Biotechnology, 1:40,000) and anti-rat (Jackson ImmunoResearch, 1:10,000) secondary antibodies, respectively, for 1 hour at room temperature. Immunoblots were developed using the Luminata Forte detection reagent (Millipore) and Hyperfilms ECL (GE Healthcare).

Total protein was extracted by using radioimmunoprecipitation assay buffer from Epstein–Barr virus-transformed BLCLs (n = 19) grown to a density of 1 × 10⁶ cells/mL. Protein extracts were quantified by using the bicinchoninic acid assay, and 25 μg of each sample was loaded on NuPAGE 4 to 12% Bis-Tris gels (Thermofisher Scientific). Western blot analysis was performed according to standard manufacturer’s protocol using nitrocellulose membranes (Thermofisher Scientific). Ponceau staining was used to evaluate equal loading and transfer. The membranes were probed with anti-tapasin rat monoclonal antibody clone 7F6 (EMD Millipore) and goat anti-rat horseradish peroxidase-linked antibody (Cytiva). Signals were detected by using enhanced chemiluminescence reagent (Cytiva) and quantified by using GeneTools software (Syngene). Western blot experiments were repeated three times. For each experiment, protein samples from 19 BLCLs were quantified and loaded on three gels, so that each gel contained at least three samples that were also loaded on a different gel. These samples were used for signal normalization across the gels. Unpaired t tests were used to determine significance.