Figure 3

Solenopsin A inhibits Akt activity (AktAR) and PDK1 activation (Lyn-PARE). NIH3T3 cells were transfected with AktAR or Lyn-PARE and serum starved for 24 h, followed by 1 h treatments with DMSO, ceramide C2, (+)-solenopsin A ((+) Sol. A), (−)-solenopsin A ((−) Sol. A) or solenopsin analogs S11-S15. To study Akt activity and PDK1 activation, 50 ng/mL of PDGF was added followed by immediate imaging. FRET ratio of regions of interest at cell cytosol and at cell periphery representing the plasma membrane were used, respectively. Akt activity is measured by AktAR response (dark grey columns) and PDK1 activation by Lyn-PARE response (light grey columns). All the ratios were normalized with the ratio before PDGF addition. Data shown are an average of at least three experiments ± s.d.