Extended Data Fig. 7. Effect of MAP4 Loss in Phosphorylation Level of EGFR and its Distribution in Endosomes.

a, b, Effect of MAP4 knockdown in endosomal localization of PI3Kα vesicles. siRNAs targeting the 3’ UTR region of MAP4 (siMAP4#3) were used to knockdown endogenous MAP4 in MDA-MB-231 cells expressing WT or the MTBD deletion mutant of MAP4. 48–72 hours post-transfection, cells were processed for immunofluorescence study using antibodies specific for endogenous p110α and clathrin or TFR. The quantification of the colocalization of p110α and CHC/TFR by Pearson’s r was shown in Figure 5c. The images shown are the representative images of multiple reproducible experiments. Scale bar, 5 μm

c, d, The phosphorylation level of EGFR in MAP4 knockdown cells. 48–72 hours post siRNA transfection, cells were stimulated with EGF before examining the phosphorylation level of EGFR by immunoblotting and immunofluorescence microscopy. The immunoblot shown is the representative images of multiple reproducible experiments. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments </p/>e, Localization of activated EGFR in endosomes in MAP4 knockdown cells. 48–72 hours post siRNA transfection for MAP4 knockdown, cells were stimulated with EGF and processed for immunofluorescence study to examine the co- localization of activated EGFR with endosomes (EEA1 and TFR) by Pearson’s r. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments Unprocessed_Western_Blots_Extended_Data_Fig7; Statistical_Source_Data_Extended_Data_Fig7