Phospho pi3k p85 tyr 458 p55 tyr199 antibody

Recombinant Human CXCL12 and anti-human CXCL12 antibody were purchased by R&D system Inc. (Minneapolis, MN, USA). LY294002 (PI3K inhibitor) was ordered from Cell Signaling Technology (Beverly, MA, USA). The monoclonal antibodies (mAbs) included PTEN antibody, phospho-PTEN (ser380) antibody, Akt antibody, phospho-Akt (ser473), PI3K p85 antibody, phospho-PI3K p85 (Tyr 458) /p55 (Tyr199) antibody were provided by Cell Signaling Technology.

After SDS-PAGE electrophoresis, wet transfer was carried out to transfer the proteins from the gel to a polyvinylidene fluoride (PVDF) (Millipore, USA) membrane using Mini Trans-Blot^® Electrophoretic Transfer Cell (BioRad, USA). The primary antibodies used were NFκB p105/50 antibody, phospho-NFκB p105 (Ser933) antibody, PI3K p85 antibody, phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody, and β-actin antibody (Cell Signaling, USA). All of the primary antibodies were diluted in 1:1000 except β-actin antibody which was diluted in a ratio of 1:1500. Chemiluminescence detection was done using ChemiDoc^TM XRS+ System (Bio-Rad, USA) and Immobilon Western Chemiluminescent HRP substrate (Millipore, USA). Image Lab^TM version 6.0 software (Bio-Rad, USA) was used for the quantification of the immunodetected protein bands. The expression of each protein was quantified and normalised against the protein expressions of their respective total protein and housekeeping gene, β-actin.