Protein a g plus agarose beads

Cells were collected, washed once with PBS, and incubated in IP lysis
buffer (25 mM Tris-HCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40,
5% Glycerol) at 4°C for 15 minutes. 300-500 μg of each
sample was then pre-cleared in Protein A/G Plus Agarose Beads (Thermo Fischer
Scientific) for one hour. Following pre-clear, samples were then incubated with
antibody-bead conjugates overnight according to manufacturer's
specifications and as mentioned previously. The samples were then centrifuged at
1000g for 1 min, and the beads were washed with 500 μL of IP lysis
buffer for five times. Proteins were eluted from the beads by boiling in
2× LDS Sample Buffer (Invitrogen) at 95°C for 5 min. Samples
analyzed by immunoblotting as previously described. Immunoprecipitation
antibodies were diluted according to manufacturer's instructions (1:200
for p53 and 1:100 for Bcl-xL).

Lipid kinase assays were performed as published (64 (link)). PI3Kγ or α isoforms were immunoprecipitated from a solution containing Protein A/G plus agarose beads (Thermo Scientific), PI3Kγ or PI3Kα antibody (diluted 1:100), and 100 μg of lysate from human lung fibroblast (19 Lu) plasma membrane fractions diluted in Triton lysis buffer as above. After washing, the beads were resuspended in 50 μl reaction buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 100 μM sodium-orthovanadate) with 10 μl of 100 mM MgCl. Then, 20 μl PtdIns (2.5 mg/ml, 50 μg total) was dissolved in TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and added to each reaction. The reactions were initiated by adding 1uL of a mixture of 10 ul 440 uM cold ATP and 10 μCi ³²P-γ-ATP, and the reactions were incubated at 25°C with continuous agitation for 10 min. The reactions were stopped by adding 20 μl 6N HCl. Lipids were extracted by adding 160 ul of 1:1 chloroform:methanol, vortexing, and centrifuging. 30 μl of the organic phase (bottom phase) was spotted onto 200 μm silica-coated TLC plates (Selecto-flexible; Fisher Scientific) pre-coated with 1% potassium oxalate. The plate was resolved via chromatography with 1:1.87 2N glacial acetic acid:1-propanol for approximately 6 hours. The plates were then dried, exposed, and lipid phosphorylation was visualized with autoradiography.