Ionomycin

T cells were sorted and seeded in a 96-well plate. Stimulating the cells with PMA (25 ng/ml) to analyze the intracellular INF-γ. Stimulating with PMA (25 ng/ml) and ionomycin (500 ng/ml) to analyze the intracellular TNF-α. Cells were stimulated with PMA (0.1 μg/ml, Sigma-Aldrich), ionomycin (0.5 μg/ml, Sigma-Aldrich) in the presence of Brefeldin A (10 μg/ml, Sigma-Aldrich) For IL-17A intracellular cytokine staining. For IL-22 production, adding IL-23 (40 ng/ml, R&D Systems, Minneapolis, MN), PMA, ionomycin, and Brefeldin A into cultures for 4 h. Cells were cultured at 37°C in DMEM medium supplemented with 20% FBS for 4 h. Following the manufacturer’s instructions, cells were fixed and permeabilized with kits for intracellular staining (eBioscience). All data were collected on a BD Fortessa LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).

For cytokine production analysis PBLs were culture for 7 days and then re-stimulated for 4 h with 15 ng/ml Phorbol-12-Myristate-13-Acetate (PMA) and 500 ng/ml Ionomycin (both from R&D systems, Minneapolis, MN) in the presence of Monensin (eBioscience, Thermo Fisher Scientific, Waltham, USA). All were added per the manufacturer’s instructions. After surface staining including rat anti-canine CD4-PE Cy7 (clone YKIX302.9) and rat anti-canine CD8α-Alexa Fluor 647 (clone YCATE55.9) (both from Bio-Rad, Hercules, USA), intracellular staining for cytokines was performed using Fixation and Permeabilization buffers (eBioscience, Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol. PBLs were fixed for 20 min in RT washed and resuspended in permeabilization buffer for 20 min followed by 30 min incubation with primary antibodies. For intracellular staining mouse anti-bovine IFN-γ-FITC antibody (clone CC302), with confirmed canine cross-reactivity was used, as well as polyclonal rabbit anti-canine IL-2 primary antibody followed by sheep anti-rabbit IgG PE conjugated secondary antibody staining (1:1,000 dilution) (all purchased from Bio-Rad, Hercules, USA). Afterwards, cells were washed with FACS buffer and analyzed using BD FACS Aria II flow cytometer (Becton Dickinson, Heidelberg, Germany).

HEK293T cells plated on glass coverslips were transfected with PKCβ^WT-YFP or β^D/A-YFP expression vectors using Lipofectamine 2000 (Life Technologies). 24 hr after transfection, the coverslips were transferred to the imaging chamber of a custom-built 2-photon laser scanning microscope system and superfused with buffer (138 mM NaCl, 1.5 mM KCl, 10 mM HEPES, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, pH 7.4) at 2 ml/min. YFP was excited at 840 nm with a Ti-Sapphire laser through a 60×, 1.1 NA water-immersion Olympus lens. A 500–550 BP emission filter was used. 512 × 512 frame scans were acquired at a rate of 1 line/4 ms, every 30 s. To stimulate translocation, the superfusion solution was switched to one containing 1 µM PDBu or 10 µM ionomycin (R&D Systems) for 15 min. The experiment was repeated three times for PKCβ^WT-YFP and twice for PKCβ^D/A-YFP, with similar results. Acquired images were exported to ImageJ and brightness/contrast was adjusted equally for all images within an experiment for display purposes.