Harvest 5 x 106 Jurkat E6.1 cells or 1 x 106 HEK293T cells transfected with vector, wild type (WT), kinase-dead mutant (K409R) and sumoylation deficient mutant (2KR [K325R, K506R]) of Myc-PKC-θ (indicated in Figure 2) by centrifugation (300 x g, 3 min, 25 °C). Wash the cells once with 1 ml ice cold PBS.Centrifuge the cells at 300 x g for 3 min at 25 °C.Add 300-500 µl lysis buffer to the cell pellets, vortex for 1-2 min.Place the tube on ice for 10 min.Centrifuge at 8,500 x g for 10 min at 4 °C.Transfer the clear supernatant to a new microcentrifuge tube.Immunoprecipitate endogenous or transfected PKC-θ by adding 2.5 µl anti-PKC-θ or anti-Myc antibodies into the clear supernatant obtained in step 7. Incubate overnight at 4 °C with rotation. Then add 30 µl protein G sepharose beads (50% [v/v] in PBS) and incubate for 2-4 h at 4 °C with rotation.Centrifuge for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant. Wash the immunoprecipitates extensively by adding 1 ml lysis buffer and vortexing for 3-5 min.Repeat step 9.Centrifuge for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant. Wash the immunoprecipitates extensively by adding 1 ml PKC-θ kinase buffer and vortexing for 3-5 min.Repeat step 11.Centrifuge for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant and resuspend the pellet in 25 μl of the reaction buffer.Incubate samples in the Thermomixer with gentle shaking at 300 rpm for 30 min at 30 °C. Stop the reaction by adding 6.25 µl 5x SDS-PAGE loading buffer. Heat samples at 95 °C for 10 min and subject the samples to 15% SDS-PAGE, then cover the gel with plastic wrap to minimize the chance of radioactive contamination. Open the exposure cassette, place the wrapped gel on the internal surface of the cassette, and then place the phosphor-storage screen on top the wrapped gel with the phosphor (white) side facing down onto the gel. Close and lock the cassette, place it for 30 min to 6 h at room temperature and then take the phosphor-storage screen out of the cassette avoiding direct light. Scan the screen with the Typhoon Trio+ system.(Optional) A pilot experiment to verify the specificity of this kinase assay is recommended. In detail, HEK293T cells were transfected with wild type Myc-PKC-θ, which has proven to be an active kinase as T538, S676, and S695 are constitutively phosphorylated on recombinant PKC-θ isolated from HEK293T expression systems (Wang et al., 2012). 24 h after transfection, Myc-PKC-θ was immunoprecipitated and subjected to the kinase assay as described previously. When doing the kinase assay, the ATP-competitive PKC-θ inhibitor or DMSO was added into the reaction buffer which served as the negative and the positive control individually.