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. 2014 Jun 1;127(11):2407–2419. doi: 10.1242/jcs.130344

Fig. 2.

Fig. 2.

Notch receptor localization shifts away from plasma membrane in BBS-depleted cells. (A) Sucrose-gradient fractionation of HEK293 cells transfected with HA-NOTCH1 alone or HA-NOTCH1+shBBS4 probed for HA, N-cadherin (plasma membrane marker), and EEA1 (endosome marker). (B) Quantification of the p180 band in A displayed as the proportion in each fraction relative to the total across all fractions. (C) Sucrose-gradient fractionation of HEK293 cells transfected with HA-NOTCH1 alone or HA-NOTCH1+shBBS1 probed for HA, N-cadherin and EEA1. (D) Quantification of p180 from C displayed as the proportion in each fraction relative to the total across all fractions. (E–G) hTERT-RPE1 cells immunostained with antibody against endogenous NOTCH1 (green). Scale bars: 10 µm. (H,I) hTERT-RPE1 cells coimmunostained for endogenous NOTCH1 (green) and plasma membrane marker N-cadherin (red) showing colocalization (arrowheads, inset). Histograms represent the intensity of green fluorescence across the white line. Arrows above histograms represent regions of plasma membrane fluorescence. Scale bars: 5 µm (J) Quantification of all intensity values within individual membrane peaks. Data represent the mean±s.d. (≥50 cells per treatment, two cross-sections per cell). *P≤0.001 compared with control (Student's t-test).