Figure 4. TIPE3 promotes the activation of PI3K-AKT and MEK-ERK pathways.

(A) Whole cell lysates were prepared from NIH3T3 cells stably transfected with TIPE3-Flag, trTIPE3-Flag, or empty vectors (left two panels), or TIPE3-Flag and/or HRasV12 vectors (right panel). Western blot (WB) was performed using antibodies against the indicated proteins. The densitometric quantification of protein signals was made using ImageJ software. Signals of cells transfected with empty vector were set as 1. (B) Whole cell lysates were prepared from NCI-H727, T24, and HT-29 cells stably expressing either shTIPE3 or shScr. Western blot was performed using antibodies against the indicated proteins. Signals of cells treated with shScr were set as 1. (C and D) NIH3T3 cells stably transfected with either TIPE3-Flag or empty vectors were serum-starved, and then stimulated with 40 ng/ml of PDGF for the indicated times, with or without the following inhibitors: LY29 (LY29004), Rapa (Rapamycin), PD98 (PD98059), Bis I (Bisindolylmaleimide I), or U-73 (U-73122). Lysates of these cells were used for Western blot with antibodies against the indicated proteins. Signals of cells transfected with vector 2.5 min after PDGF stimulation were set as 1. (E) Cellular levels of PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ in NIH3T3 cells stably transfected with either TIPE3-Flag or empty vector were estimated by protein-lipid overlay assay with GST-PLCδ-PH and GST-GRP1-PH domains as described in Supplemental Experimental Procedures. Signals of cells transfected with empty vector were set as 1. Y-axis values represent means ± SD; ns denotes not significant; * p<0.05, and ** p< 0.01. p- indicatedes phosphorylated. The experiments were performed at least three times with similar results. See also Figure S4.