Ms safe protease and phosphatase inhibitor cocktail

Briefly, crushed frozen brains were added to a lysis buffer containing 20 mM MOPS, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM PMSF, and 10 μg/mL leupeptin/aprotinin, 50 mM β-glycerolphosphate, 50 mM sodium fluoride, and 1 mM sodium vanadate. Benzamidine, leupeptin/aprotinin, β-glycerolphosphate, sodium fluoride, and sodium vanadate were supplied via the reconstitution of a lyophilized powder (i.e. MS-SAFE Protease and Phosphatase Inhibitor Cocktail [Sigma-Aldrich]). Samples were homogenized three times on ice for 20, 10, and 10 seconds, then sonicated for 10 seconds on ice. Samples were subsequently centrifuged at 15,000xg for 30 minutes at 4°C. Protein concentration in the supernatant was determined using the Pierce BCA Protein Assay (Life Technologies) and an equal concentration of protein from each sample was used for the assay. The p70S6 kinase activity assay was carried out according to the manufacturer’s instructions (Enzo Life Sciences, ADI-EKS-470).

The stimulation method of J.OT1 cells is based on a previously established study^{21 (link)}. For each replicate per condition, 15 million J.OT1 cells were washed and resuspended in serum-free RPMI at a concentration of 50 million cells per mL. Cells were then incubated with either 10 nM of H-2K^b MHC tetramer (Class I, human beta-2-microglobulin) or ~1 ug/mL of anti-CD3 IgM antibody C305 on ice for 1 h. C305 was diluted in Dulbecco’s phosphate-buffered saline (DPBS) prior to stimulation. MHC tetramers^{30 (link)} were synthesized by the NIH Tetramer Core Facility (Atlanta, GA) using custom peptides from GenScript. The peptide sequences are SIINFEKL (OVA), SIITFEKL (T4), SIIGFEKL (G4), and RGYVYQGL (VSV). Stimulation was initiated by transferring the cells from ice to 37°C. After 3 mins of stimulation at 37°C, cells were lysed with equal volume of lysis buffer (pH 7.6) containing 1% (w/v) sodium dodecyl sulfate (SDS), 100 mM Tris-HCl and 1X MS-SAFE Protease and Phosphatase Inhibitor cocktail (Sigma-Aldrich MSSAFE). PV treatment was performed by incubating J.OT1 cells with 500 μM PV (prepared by mixing equal volume of 1 mM sodium orthovanadate and 1 mM hydrogen peroxide) for 10 minutes at 37°C and lysed with the lysis buffer as mentioned.