Fig. 3 |. LT affects PI3K signalling.

a, LT inhibition of Akt activation. Akt phosphorylation was assessed in HeLa cells treated with LT (1 μg ml⁻¹) or the MEK1 inhibitor PD98059 (50 μM) (PD) for 5 h or 19 h, and stimulated with 1 mM H₂O₂ (20 min). LT inhibits Akt activation and mTOR signalling. b–e, C57BL/6J BMDMs were treated with the MEK1 inhibitors PD98059 (50 μM) or trametinib (Tr) (200 nM), the p38 pathway inhibitors PH-797804 (PH) (50 nM) or SB239063 (SB) (100 nM), and/or the PI3K pathway inhibitors wortmannin (Wm) (1 μM), LY924002 (LY) (50 μM), and LT or LT(W271A) (1 μg ml⁻¹) for 6 h, 12 h or 24 h, and stimulated with LPS (1 μg ml⁻¹, 60 min), insulin (200 nM, 45 min) (Ins) or H₂O₂ (1 mM, 30 min) before analysis by western blot. In b, inhibitors were also added at 100 nM 1 h before LPS stimulation. LC indicates loading control, antibody cross-reactive species that indicate equal protein loading in each lane. f, LT does not induce PTEN dissociation from p85α. BMDM lysates were treated with LF (1, 10 or 100 μg ml⁻¹) for 5 h at 37 °C before immunoprecipitation with p85α antibody and assessment of PTEN association by western blot (WB). WC, whole-cell lysate; IgG, non-specific rat antibody control. g, LT cleavage does not result in PTEN destabilization. HT1080 cells and C57BL/6J BMDMs were treated with LT (1 μg ml⁻¹) for the indicated time before western blot with PTEN antibody. Blots are representative of two to six similar experiments. Densitometry of pAkt bands is presented in Supplementary Table 1.