Anti pkcθ clone 27 pkcθ

Proteins were extracted in 50 mM Tris, 120 mM NaCl, 1 mM EDTA with 1% IGEPAL CA-630 and protease and phosphatase inhibitor cocktails (Thermo) before SDS-PAGE and western blotting. The antibodies used in this study were anti-PKCθ clone 27/PKCθ (BD Bioscience), anti-PKCθ pT538 ployclonal (Cell Signaling Technologies #9377) , anti-c-Jun Rabbit monoclonal (Cell Signaling Technologies #9165), anti-c-fos Rabbit monoclonal (Cell Signaling Technologies #2250), anti-fosB Rabbit monoclonal (Cell Signaling Technologies #2251), anti-JunB Rabbit polyclonal (Santa Cruz Biotehnology #sc-46X) anti-JunD Rabbit polyclonal (Santa Cruz Biotehnology #sc-74X) and anti-beta-2-microglobulin (Abcam #AB75853).

Before imaging, BM-DC were incubated with 1 μg/ml LPS (Sigma) for 16-18 hours, washed and then incubated for 2 hours with 1 μg/ml MBP Ac1-9[4Y]. DC-T cell couples for confocal microscopy were prepared by combining activated and peptide-loaded BM-DC and magnetically enriched CD4+ T cell at a ratio of 1:2 at 3x10⁶ cells/ml. Coupling was synchronized by brief centrifugation (75 g) in a round bottom plate and the cells were allowed to interact for 5 minutes at 37°C before the cells were gently transferred to pre-warmed glass slides and incubated for a further 5 minutes. Cells were fixed in 4% buffered PFA before immune-labeling. For TIRF-M imaging, glass coverslips (Mat-Tek II) were pre-coated with 2 μg/ml anti-CD3ε clone 145-2C11, BD) and 1 μg/ml anti-CD28 (clone 37.51, BD). CD4⁺ T cells were allowed to interact with the antibody coated surface for 8 minutes at 37°C before fixation and immune-labeling. Primary antibodies used were anti-PKCθ clone 27/PKCθ (BD Bioscience) at 2.5 μg/ml, anti-Zap70 clone D1C10E (Cell Signaling Technologies) at 2.5 μg/ml, anti-CD28 clone PV.1 (Abcam) at 1 μg/ml and anti-Cbl-b polyclonal H121 (Santa Cruz - discontinued) at 5 μg/ml. Secondary antibodies, goat anti-rabbit IgG-DyLight488, donkey anti-mouse IgG-Cy3 and goat anti-Armenian hamster-Cy2. (Jackson Immunoresearch) were used at 2.5 μg/ml.