JCS036038 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Folate receptor (FR) is localized on the apical domain of the plasma membrane of MDCK cells. MDCK cells stably expressing FR, grown on filter for 4 days were stained using anti-FR antibody (Alexis Biochemicals, Lausen, CH), followed by a TRITC-conjugated secondary antibody in non-permeabilised conditions. Serial confocal sections were collected from the top to the bottom of cell monolayers. Bars, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Cholesterol addition does not alter the integrity of monolayer and does not affect the polarity of two endogenous proteins. Cholesterol (10 mM) was added to MDCK cells grown on filters for 30 or 60 minutes at 37°C. (A) Medium was replaced and transepithelial resistance (TER) was measured periodically over 60 minutes in the absence (control) or in presence of cholesterol (+chol) added to both sides. (B) Cells were fixed and stained using specific antibodies against GP114 and Na⁺/K⁺-ATPase under non-permeabilising and permeabilising conditions, respectively. Serial confocal sections were collected from the top to the bottom of cell monolayers. Bars 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Cholesterol addition does not alter the unpolarised distribution of the double cysteine GFP-FR mutant (S49/71). Cholesterol (10 mM) was added to MDCK cells stably expressing the double cysteine GFP-FR mutant (S49/71) grown on filters for 30 minutes at 37°C. Then cells were fixed and stained with an anti-Myc antibody (from Santa Cruz Biotechnology, CA) followed by a TRITC-conjugated secondary antibody under non-permeabilising conditions. Serial confocal sections were collected from the top to the bottom of cell monolayers. Bars, 10 µm. Mean fluorescence intensities at the apical and basolateral domains were measured and expressed as percentages of total fluorescence.