Figure 6: MAP4 is Required for PI3,4,5P₃ Generation.

a, b, MAP4 knockdown impairs EGF stimulated PI3,4,5P₃ generation. Three different siRNAs were used individually to knockdown MAP4 in MDA-MB-231 cells. EGF induced PI3,4,5P₃ was analyzed by immunofluorescence microscopy and MAP4 knockdown by immunoblotting. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

c, d, Rescue of EGF induced PI3,4,5P₃ generation. 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 induced PI3,4,5P₃ was analyzed by immunofluorescence microscopy. The knockdown of endogenous MAP4 and expression of ectopically expressed wild type or mutant form of MAP4 were analyzed by immunoblotting. Scale bar; 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

e, Effect of MAP4 knockdown in PI3Kα mutant expressing cells (Cal51) and EGFR overexpressing cells (A431). siRNA was used to knockdown endogenous MAP4 (siMAP4#1) and the effect on PI3,4,5P₃ was analyzed by immunofluorescence microscopy. The knockdown of MAP4 was examined by immunoblotting. Scale bar, 5 μm; Error bars denote mean±SD, n=30 cells from representative experiments

f, Effect of MAP4 knockdown in PI3,4,5P₃ generation in endosomes. 48–72 hrs post-transfection with siRNA for MAP4 knockdown, cells were lifted and allowed to adhere to coverslips coated with Col.I for 30 minutes. Then, cells were immunostained with antibodies specific for PI3,4,5P₃ and endosomal markers (EEA1 and TFR). PI3,4,5P₃ co-localized with endosomes were analyzed by immunofluorescence microscopy. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

g, Analysis of EGF induced PI3,4,5P₃ generation in MAP4 knockdown cells by subcellular fractionation. siMAP4#3 was used to knockdown endogenous MAP4 in MDA-MB-231 cells or its transfectants expressing WT or MTBD deletion mutant of MAP4. 48–72 hrs post-transfection, cells were stimulated with EGF for 5 minutes and harvested for subcellular fractionation. The PI3,4,5P₃ generated in plasma membrane vs. endosomal fractions were analyzed by an ELISA assay as indicated in “Method”. Error bars denote mean±SD; n=3 independent experiments

Unprocessed_Western_Blots_Fig6; Statistical_Source_Data_Fig6