Express protein labeling mix

Naive T cells cultured for 3 days were starved for 30 min in methionine and cysteine-free RPMI medium (Cellgro). The cells were labeled with [³⁵S] methionine/[³⁵S] cysteine Labeling Mix (Express Protein Labeling Mix, Perkin Elmer) for 1 h at a final concentration of 50 µCi/ml. After the labeling, cells were washed twice with cold PBS and nuclear extract were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). Immunoprecipitation of Bcl6 was performed using a Bcl6 antibody (4242, Cell Signaling Technology). Bcl6 immunoprecipitates were separated by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was dried and placed on a film for autoradiography at −80 °C.

Cells were starved in methionine- and cysteine-free DMEM for 30 min at 37°C and then pulsed in the same media supplemented with ³⁵S-labeled methionine and cysteine (Express Protein Labeling Mix, Perkin Elmer) at 200 μCi/ml for 15 min at 37°C. Cells were then washed with ice-cold RF10 and then incubated in RF10 at 37°C. At the selected timepoints, cells were washed in PBS and frozen. Cell pellets were lysed in 0.5% IGEPAL CA-630 (Sigma-Aldrich), 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂with Complete Protease Inhibitor Cocktail (Roche), and centrifuged 13,000 × g for 10 min to separate nuclei. Lysates were precleared twice with normal mouse serum (Sigma-Aldrich) and protein G–Sepharose and twice with protein G–Sepharose alone. MHC-I was immunoprecipitated using w6/32 antibody and protein G–Sepharose, and the immunoprecipitates were washed in NET buffer (0.5% IGEPAL CA-630, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA) three times. Precipitates were treated with Endoglycosidase Hf (New England Biolabs) according to the manufacturer's instructions. Proteins were denatured in reducing LDS-PAGE sample buffer and separated on NuPAGE 4–12% Bis-Tris precast gels (Life Technologies) before transferring onto PVDF membranes using the iBlot system (Life Technologies). Membranes were dried and exposed to a storage phosphor screen (GE Healthcare) and imaged on a Typhoon imager (GE Healthcare).