Detection of calcineurin and mTOR activities

Calcineurin phosphatase activity was detected by Calcineurin Cellular Activity Assay Kit from Millipore (Billerica, MA) and mTOR signaling was detected by Calcium Detection Kit from Abcam (Cambridge, MA). The manufacturer’s protocols were followed. Briefly, Jurkat cells (2 million cells/mL) were lysed using lysis buffer. For calcineurin activity, the cell lysates were desalted by gel filtration to remove free phosphates before the assay and were subjected to calcineurin activity assay. The absorbance of each sample was read at 620nm using the plate reader. For mTOR ELISA assay, the cell lysates were added to each well and antibody against phosphorylated mTOR at serine 2448 was used to detect the activity of mTOR signaling. The absorbance of each sample was read at 450nm using the plate reader.