Determining the effects of three screened compounds on membrane PIP3 and PTEN levels.
(A) Tat-expressing CHME5 cells were treated with and without LPS/CHX in the presence of the three different drug compounds at 20 µM for 20 minutes. Cell lysates were prepared for determining the membrane PIP3 level. Data were calculated by generating a standard curve of known concentrations for PIP3 provided from the manufacturer (Data S2). Control (Con), no treatment; LPS, 50 µg/ml LPS and 10 µg/ml cycloheximide; Lancemaside A1 (LA), Arctigenin (AR), Compound K (CK) and Wortmannin (WM). (B) Tat-expressing CHME5 cells were treated in the presence or absence of LPS/CHX and 5, 10, and 20 µM of each drug for 20 minutes. Cellular membrane fractions were processed and examined by western blot analysis for PTEN. β-actin was used as a loading control. The fold changes in the PTEN level were determined as described in methods.