Pi3 kinase activity elisa kit

T cells were washed with ice-cold PBS and lysed on ice for 30 minutes in RIPA buffer containing protease inhibitor. Cell lysates were immunoprecipitated with the appropriate antibodies using protein A/G agarose beads. Samples were then used for immunoblot analysis with the indicated antibodies. PI3K activity was detected using the PI3-Kinase Activity ELISA Kit (K-1000s, Echelon) according to the manufacturer’s instructions.

p110α activity were measured by PI3-Kinase activity ELISA Kit (Echelon) according the manufacture protocol with some modifications. After treatment, NRCMs were lysed with ice-cold buffer containing 137 mM NaCl, 20 mM Tris pH7.4, 1 mM Cacl2, 1 mM Mgcl2, 1% Nonidet P-40, phosphatase inhibitor cocktail (Roche) and protease inhibitor cocktail (Roche) and solubilized by continuous stirring for 30 min at 4°C. After centrifugation (16,000 g, 15 min), the supernatant was collected, and 800 mg of homogenate (in 600 μl) was immunoprecipitated with p110α antibody from santacruz (H-201). After 4hr incubation at 4°C, Protein A-Dyna magnetic Beads (Invtrogen) was added, and the immune complex was washed four times with buffer (100 mM NaCl, 1 mM Na3VO4, and 20 mM HEPES, pH 7.5) and resuspended in 40 μl of KBZ buffer (180 mM NaCl-20 mM HEPES, pH 7.5). The kinase reaction was run for 3 hr at room temperature. Converted PIP3 level from PIP2 was measured by Virtor3 at 450 nM.

PI3‐Kinase biochemical activity towards ITPP, BPBPP and IHP was assessed by an ELISA competition assay. Class I phosphoinositide 3‐kinase (PI3‐K) activity is measured through their reaction product, PtdIns(3,4,5)P₃ (PIP3) using the PI3‐Kinase activity ELISA kit (Echelon. K‐1000 S). PI3‐K reactions are run with PI(4,5)P₂ (PIP2). The enzyme, the PIP3 standards, and the controls were mixed and incubated with highly specific PIP3 binding and then transferred to a PIP3‐coated microplate for competitive binding with the PIP3 detector protein, further detected by a peroxidase‐linked secondary detector and quantitatively evidenced by a colorimetric reaction to estimate the amount of PIP3 produced. The quantitative colorimetric signal at 560 nm was inversely proportional to the amount of PI(3,4,5)P₃ produced by PI3K. Recombinant PI3K was allowed to react with PI(4,5)P₂ (5 × 10⁻⁶ M) to produce PI(3,4,5)P₃ in the presence or not of ITPP, BPBPP or IHP (concentration range 0.1 × 10⁻⁶ M to 100 × 10⁻⁶ M).

The PI3Kα inhibitory activity of Z86 was performed as previously with the PI3-Kinase Activity ELISA kit (K-1000s, Echelon) based on the production of PIP3 [36 (link)].

PI3K activity was measured using a PI3-Kinase Activity ELISA Kit from Echelon Biosciences, following manufacturer’s instruction. Briefly, PI3K was pulled down from whole cell lysates using an antibody for PI3K/p85. Each sample was prepared from approximately 1 × 10⁶ cells. The entire immunoprecipitate from each sample was mixed with 30 μl of KBZ reaction buffer, which was then mixed with 30 μl of 10 μM PI(4,5)P2 substrate and incubated for 2 h at 37 °C. The kinase reaction was terminated by adding 90 μl of kinase stop solution to each reaction solution, and 60 μl of each mixture was transferred together with 60 μl of PIP3 detector to a well in the incubation plate. After incubation at RT for 1 h, 100 μl per sample from the incubation plate was transferred to the detection plate and incubated for 1 h at RT. The detection plate was washed with TBST, incubated with the HRP-conjugated secondary detector for 30 min, washed again with TBST, and the immobilized HRP was measured by a standard colorimetric assay, using 3,3′,5,5′-tetramethylbenzedine as a substrate and a Synergy 2 Multi-Mode Microplate Reader to record absorbance.