Fig. 6
From: NUAK1 coordinates growth factor-dependent activation of mTORC2 and Akt signaling
NUAK1 is localized at early-endosomes but not at the PM, lysosomes, and late-endosomes. A IB of NUAK1 subcellular location. Subcellular fractionation of the membrane and cytoplasmic fractions of MDA-MB-231 cells after 0, 10 and 30 min of EGF stimulation. p-Akt S473 was used as a positive control. RalA was used as a control for the membrane fraction. GAPDH was used as a control for the cytoplasmic fraction. B, C Immunofluorescence (IF) of MDA-MB-231 cells after 0 (−EGF) and 15 (+EGF) minutes of EGF stimulation. B Left, Representative confocal images for FLAG-NUAK1 WT and EGFR-GFP, arrows indicate the distance (µm) analyzed; Right, Histogram of the fluorescence intensity profile across the arrow for both red and green channels. Red, FLAG-Tagged-NUAK1; Green, EGFR-GFP (PM marker); Blue, nuclei. C Representative confocal images for FLAG-NUAK1 WT and Lamp1. Green, FLAG-Tagged-NUAK1; Red, endogenous Lamp1 (lysosome marker); Blue, nuclei. D Quantification of FLAG-NUAK1/Lamp1 co-localization from C. Each bar represents the mean ± SD, not significant (ns), Student t test. E IF of MDA-MB-231 cells after 0 (−EGF) and 30 (+EGF) minutes of EGF stimulation. Left, Representative confocal images for FLAG-NUAK1 WT and Lamp1-YFP, arrows indicate the distance (µm) analyzed; Right, Histogram of the fluorescence intensity profile across the arrow for both red and green channels. Red, FLAG-Tagged-NUAK1; Green, Lamp1-YFP (lysosome marker); Blue, nuclei. F IF of MDA-MB-231 cells after 0 (−EGF) and 15 (+EGF) minutes of EGF stimulation. Left, Representative confocal images for FLAG-NUAK1 WT and Rab7-GFP, arrows indicate the distance (µm) analyzed; Right, Histogram of the fluorescence intensity profile across the arrow for both red and green channels. Red, FLAG-Tagged-NUAK1; Green, Rab7-GFP (late-endosome marker); Blue, nuclei. G IF of MDA-MB-231 cells after 0 (−EGF) and 30 (+EGF) minutes of EGF stimulation. Representative confocal images for FLAG-NUAK1 WT and mRFP-Rab5. Green, FLAG-Tagged-NUAK1; Red, mRFP-Rab5 (early-endosome marker); Blue, nuclei. H Quantification of FLAG-NUAK1/mRFP-Rab5 co-localization from G. Each bar represents the mean ± SD, not significant (ns), Student t test