shRNA-driven PI5KIα and PI5KIγ silencing in NK92 cells. (A) Analysis of PI5KI isoform expression in NK cells. Total RNA was extracted from NK92 cells, freshly isolated NK cells, and HeLa cells and subjected to RT-PCR with PI5KIα, PI5KIβ, and PI5KIγ; 35 (α and γ) or 40 (β) cycles of RT-PCR analysis is shown. GAPDH-specific PCR was used as loading control. (B) NK92 cells were infected with lentiviruses encoding shRNA sequences targeting PI5KIβ (shRNA-ctr), PI5KIα (shRNA-PI5KIα), or PI5KIγ (shRNA-PI5KIγ). Total cell lysates of infected populations and HeLa cells were analyzed by immunoblotting with the indicated Abs. (C) Total phospholipids were extracted from 32P-radiolabeled uninfected (NI) or silenced NK92 cells. Equal counts of lipids were resolved by TLC followed by autoradiography (top). The spot corresponding to PIP2 was quantified by densitometric analysis. PIP2 levels of uninfected population were assumed as 100%. Data represent mean plus or minus SD of 3 independent experiments (bottom).
