Pten activity elisa

After 22 h conditioning with varying doses of GSE, The PTEN Activity ELISA was used to quantify the effects of GSE on phosphatase activity of PTEN by detection of the product, PIP₂, per the manufacturer’s instructions (Echelon Biosciences Inc. Salt Lake City, UT). Briefly, conditioned cells were lysed in lysis buffer followed by immunoprecipitation. PTEN were then diluted in the PTEN reaction buffer and incubated using PI(3,4,5)P₃ as a substrate. After the PTEN reactions were complete, reaction products were added to the PI(4,5)P₂-coated microplate and a PI(4,5)P₂ detector protein was then added for competitive binding. A peroxidase-linked secondary detector and colorimetric detection was used to detect PI(4,5)P2 detector binding to the plate. The colorimetric signal was inversely proportional to the amount of PI(4,5)P₂ produced by PTEN.

Following stimulation (as described, e.g. 5 min with VEGF or VEGF+C3ar1-A/C5ar1-A) cells were lysed and CK2 or PTEN were IP’d overnight at 4°C. CK2 activity was assayed by measuring transfer ³²P to a CK2 substrate peptide via Millipore’s Casein Kinase 2 Assay Kit (Cat#17–132) per the manufacturer’s instructions. PTEN Activity in IPs was determined using Echelon’s (Salt Lake City, UT) PTEN Activity ELISA.