JCS017301 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Normal EGFR degradation is delayed in cells transfected with siRNA against RILP using two alternative siRNA duplexes. Control cells or RILP siRNA-treated cells were pretreated with 10 g/ml cycloheximide for 1 hour to prevent synthesis of new proteins. The cells were treated with 50 ng/ml EGF and harvested after 15, 60, 120 or 180 minutes. The remaining EGFR expression in EGF-treated cells was determined by western blotting, using β-tubulin to verify equal loading. The experiment was repeated two times and a representative membrane is shown.

• Supplemental Figure S2 -

  Fig. S2. EGFR degradation assayed by confocal microscopy is reduced in RILP-depleted cells. (A) HeLa cells transfected with control RNA or two alternative RILP siRNA duplexes were grown on coverslips. The cells were pretreated with 10 μg/ml cycloheximide (1 hour) before they were stimulated with 50 ng/ml EGF for 15, 60, 120 or 180 minutes. The cells were permeabilized before fixation and stained with antibody against EGFR. (B) All the images were scanned at the same settings below saturation and the EGFR intensity at each time point was quantified using the Zeiss LSM 510 software (version 3.2). The graph presented shows the average of three separate experiments (±s.e.) in which at least 15 cells were quantified per experiment.