FIGURE 7: MAP4 is Required for PI3K/Akt Signaling.

a, MAP4 loss impairs activation of Akt downstream of EGFR. Three different siRNAs individually used to knockdown MAP4 in MDA-MB-231 cells followed by examination of EGF induced Akt activation by immunoblotting. Error bars denote mean±SD; n=3 independent experiments

b, The activation level of Akt in MAP4 knockdown cells at different time points. 48–72 hrs post-siMAP4#1 transfection, the activation level of Akt were examined after EGF stimulation at different time points. Error bars denote mean±SD; n=5 independent experiments

c, d, e, Rescue of EGF induced Akt activation in MAP4 knockdown cells. siMAP4#3 was used to knockdown endogenous MAP4 in MDA-MB-231 cells expressing WT or the MTBD deletion mutant of MAP4. 48–72 hrs post-transfection, cells were stimulated with EGF for 5 minutes and activation level of Akt examined by immunoblotting or immunofluorescence microscopy. The image shown is the representative of multiple reproducible experiments. Scale bar, μm; Error bars denote mean±SD; n=4 independent experiments (c), n=30 cells from representative experiments (e)

f, Analysis of EGF induced Akt activation in MAP4 knockdown cells by subcellular fractionation. siMAP4#3 was used to knockdown endogenous MAP4 in MDA-MB-231 cells expressing WT or the MTBD deletion mutant of MAP4. 48–72 hrs post-transfection, cells were stimulated with EGF and harvested for subcellular fractions followed by examination of activated Akt in plasma membrane vs. endosomal fractions by immunoblotting. The data shown is the representative of multiple experiments.

g, MAP4 knockdown impairs the activation level of Akt in different cell types. MDA-MB-468, HCT116, SCC-1, and HeLa cells were transfected with siMAP4#1 or siMAP4#2. The cells were harvested 72 hrs-post siRNA transfection and activation level of Akt examined by immunoblotting. Error bars denote mean±SD; n=5 independent experiments

h, MAP4 knockdown impairs the activation level of Akt in MDA-MB-231 cells ectopically expressing the wild type or mutant form of p110α. The cells were transfected with siMAP4#1 and harvested 72 hrs-post transfection to examine the activation level of Akt by immunoblotting. Error bars denote mean±SD; n=4 independent experiments

Unprocessed_Western_Blots_Fig7; Statistical_Source_Data_Fig7