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, .
{"action": "wash", "output": "", "temperature": ["ice cold"], "force": ["300 x g"], "reagent": ["PBS"], "time": ["3 min"]}
C.Transfer the clear supernatant to a new microcentrifuge tube
{"action": "transfer", "output": "the clear supernatant", "container": ["new microcentrifuge tube"]}
Incubate overnight at 4 °C with rotation.
{"action": "incubate", "output": "", "temperature": ["4 \u00b0C"]}
Then add 30 µl protein G sepharose beads (50% [v/] in PBS) .
{"action": "add", "output": "", "volume": ["30 µl"], "reagent": ["protein G sepharose beads (50% [v/] in PBS)"]}
incubate for 2-4 h at 4 °C with rotation #####%%%%% output "the immunoprecipitates" are used in line12
{"action": "incubate", "output": "the immunoprecipitates", "time": ["2-4 h"], "temperature": ["4 \u00b0C"]}
Wash the immunoprecipitates extensively by adding 1 ml lysis buffer for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant. #####%%%%% output "the supernatant" are not used
{"action": "wash", "output": "the supernatant", "reagent": ["the immunoprecipitates"]}
Wash the immunoprecipitates extensively by adding 1 ml PKC-θ kinase buffer for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant in 25 μl of the reaction buffer. #####%%%%% output "the supernatant" are not used
{"action": "wash", "output": "the supernatant", "reagent": ["the immunoprecipitates"]}
Stop the reaction by adding 6.25 µl 5x SDS-PAGE loading buffer. #####%%%%% output "the reaction" are not used
{"action": "stop", "output": "the reaction", "reagent": ["SDS-PAGE loading buffer"]}
Heat samples at 95 °C for 10 min , then cover the gel with plastic wrap to minimize the chance of radioactive contamination. #####%%%%% output "radioactive contamination" are not used
{"action": "cover", "output": "radioactive contamination", "temperature": ["95 \u00b0C"], "time": ["10 min"]}
Open the exposure cassette, .
{"action": "open", "output": "", "device": ["exposure cassette"], "concentration": [""]}
place the wrapped gel on the internal surface of the cassette,
{"action": "place", "output": "", "container": ["the wrapped gel", "the cassette"]}
then place the phosphor-storage screen on top the wrapped gel with the phosphor (white) side facing down onto the gel
{"action": "place", "output": "", "device": ["the phosphor-storage screen"]}
Close place it for 30 min to 6 h at room temperature .
{"action": "place", "output": "", "time": ["30 min"], "temperature": ["room temperature"]}
then take the phosphor-storage screen out of the cassette avoiding direct light #####%%%%% output "direct light" are used in line36
{"action": "take", "output": "direct light", "device": ["the phosphor-storage screen"], "volume": [""]}
Scan the screen with the Typhoon Trio+ system. #####%%%%% output "the screen" are not used
{"action": "detect", "output": "the screen", "device": ["the Typhoon Trio+ system"]}
(Optional) A pilot experiment to verify the specificity of this kinase assay is recommended. #####%%%%% output "recombinant PKC-θ" are used in line36
{"action": "advise", "output": "recombinant PKC-\u03b8", "device": ["", ""]}
In detail, HEK293T cells were transfected with ., 2012.

wild type Myc-PKC-θ, which has proven to be an active kinase as T538, are constitutively phosphorylated on recombinant PKC-θ isolated from HEK293T expression systems (Wang et al
{"action": "activate", "output": "", "reagent": ["T538", "recombinant PKC-\u03b8", "direct light"]}
24 h after transfection, Myc-PKC-θ was immunoprecipitated .

subjected to the kinase assay as described previously #####%%%%% output "the kinase assay" are not used
{"action": "conduct", "output": "the kinase assay", "device": [""]}
When doing the kinase assay, the ATP-competitive PKC-θ inhibitor was added into the reaction buffer which served as the negative individually. #####%%%%% output "the kinase assay" are not used
{"action": "add", "output": "the kinase assay", "reagent": ["ATP-competitive PKC-θ inhibitor"]}