Fig. 2.

Reduced amounts of NOTCH1^G661S and NOTCH1^A683T are presented at the plasma membrane. A. Proteins expressed on the plasma membrane of NIH3T3 stable cell lines expressing either rNOTCH1^WT, rNOTCH1^G661S, or rNOTCH1^A683T receptor were labeled by biotinylation. Ten percent of the total protein extract was removed (T) after which the remaining lysate was subjected to streptavidin pull down (B), to isolate cell-surface proteins. Western blot analysis of total protein lysate (T) and cell-surface proteins (B) was performed using an anti-HA antibody. The 180 kDa band present in all biotinylated fractions indicates that only the S1 cleaved form of rNOTCH1 reaches the plasma membrane efficiently. Note the relative decrease in biotinylated receptor in cell lines expressing rNOTCH1^G661S or rNOTCH1^A683T mutants compared to cell lines expressing wild-type NOTCH1. anti-GM130 antibody was utilized to assess fraction purity. Representative data are shown. B. Band intensities were quantified and the ratio of biotinylated NOTCH1 to total NOTCH1 was calculated. Results are graphed as mean±SD from three independent experiments. p-values were calculated using ANOVA followed by Bonferroini post hoc.