Supplemental Data Arabidopsis Sterol Endocytosis Involves Actin-Mediated Trafficking via ARA6-Positive Early Endosomes

Supplemental Experimental Procedures Confocal UV Laser Scanning Microscopy of Filipin-Sterol Fluorescence Dot-Blot Detection of Filipin-Sterol Fluorescence Analysis of Arabidopsis roots was performed by employing a Nikon Sterols (Sigma) and 4-cholesten-3-one (Sigma) in chloroform at 20 Diaphot 300 inverted microscope, connected to a Nikon RCM 8000 mM were spotted onto Transblot nitrocellulose membranes (BioRad) confocal laser scanning unit (Nikon) equipped with an Argon laser within a range from 1 ng to 5 g. Since filipin-sterol complexes for UV excitation of filipin-sterol complexes at 351 nm and a Argonadsorb light in the UV range and emit fluorescence at 480 nm [S1], Krypton laser for 488/568 nm excitation of FITC, GFP, YFP, Alexa blots were overlaid with 200 g/ml filipin III in MTSB and were 488, or FM4-64. Fluorescence emission was observed by using apobserved for fluorescence under UV-B excitation at 312 nm by empropriate filter sets. Sequential scans were taken under UV and 488/ ploying Image Master (Pharmacia Biotech). Cholesterol oxidase 568 nm excitation, to avoid bleed-through of filipin-sterol fluores(COX) catalyzes isomer-specific oxidation of 3-hydroxysterols, cence into the green/yellow-red channels. In all single, double, and including several plant sterols [S2]. For COX treatments, sterol dottriple labeling experiments, appropriate controls were included to blots were blocked with 3% bovine serum albumin (BSA, Sigma) in distinguish signals from background fluorescence in control roots, COX buffer (COB: 50 mM sodium phosphate buffer [pH 7.2]) for 30 and to ensure that no bleed-through occurred between channels. min. Blots were washed once in COB, overlaid with COX (2 U/ml per 10 cm) from Nocardia erythropolis (Roche Diagnostics), or the same amount of heat-denatured enzyme in COB, and incubated for 3 hr at 37 C. Blots were washed once with demineralized water (demin H2O), once in MTSB, incubated with 200 g/ml filipin III in MTSB for 15 min, and observed for fluorescence.