Figure 5
Reduced surface expression of Notch receptors in Tm2d3-deficient cells. (a) Reduced expression of Notch proteins in Tm2d3-deficient cells. Primary cultured embryonic fibroblasts from wild type (+ / +) or Tm2d3-deficient (−/−) littermate embryos were analyzed with immunoblotting with the indicated antibodies. (b) Reduced expression of Notch1 FL, Notch1 ECD, and Notch2 ECD in Tm2d3-deficient cells. Two rounds of independent experiments were additionally conducted as shown in (a). Adjusted volumes for the bands for Notch1 FL, Notch1 ECD, and Notch2 ECD in each extract were normalized to that of the vinculin band in the corresponding lane. In each experiment, the value in the + / + cells was set as 1, and the value for the −/− cells was calculated relative to this amount. The error bars indicate the standard deviations (n = 3). *P = 3.2 × 10–2 using Welch’s t-test. **P = 2.4 × 10–2 using Welch’s t-test. ***P = 2.8 × 10–2 using Welch’s t-test. Notch2 FL was barely detectable in all the three rounds of experiments. (c) mRNA expression levels for Notch1, Notch2, and Tm2d3 in Tm2d3-deficient cells. Three sets of the primary cultured embryonic fibroblasts of the indicated genotypes from littermate embryos were subjected to RNA isolation and real-time PCR analysis using the indicated primers. As the calculations shown in c, the value in the + / + cells was set as 1, and the value of the −/− cells was calculated relative to this amount in each of the sets. The error bars indicate the standard deviations (n = 3). *P = 7.0 × 10–8 using Welch’s t-test. (d) Reduced surface biotinylation of Notch receptors in Tm2d3-deficient cells. Primary cultured embryonic fibroblasts of the either genotype ([+ / +], [−/−]) isolated from littermate embryos were incubated with a non-membrane permeable biotinylation reagent. Immunoblotting was done for proteins fractionated with an avidin agarose with the indicated antibodies. In the anti-Notch1 ECD blot, the band above 250 kDa is non-specific. Notch1 FL is not efficiently transported to the cell surface and biotinylated. As the anti-Notch2 ECD antibody (sc-518169) did not clearly react with the biotinylated antigen, another antibody against Notch2 ECD (sc-5545) was used for this analysis. The murine epitope of the sc-518169 antibody contains a lysine residue (Lys268) that can be biotinylated. (e) Reduced surface immunoreactivity of Notch1 ECD in Tm2d3-deficient cells. Primary cultured embryonic fibroblasts of either genotype ([+ / +], [−/−]) isolated from littermate embryos were incubated with the indicated anti-Notch1 antibody followed by fixation, visualization by secondary antibodies, and confocal microscopy. Vesicles stained represent endocytic vesicles that contain the first antibody, as the incubation with the first antibody was performed at room temperature (see Methods).
