QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 1. siRNA-mediated reduction of clathrin inhibits agonist-induced endocytosis of LPA₁. (A) Stably-transfected LPA₁/HeLa cells were incubated in the presence or absence of 10 µM LPA for 30 minutes, fixed and processed for immunofluorescence detection of FLAG-tagged LPA₁ with M1 mouse anti-FLAG antibodies and fluorescently-labeled secondary antibodies. (B) Cell lysates were prepared from stably-transfected LPA₁/HeLa cells, which were either mock transfected (siControl) or transfected with clathrin siRNA (siClathrin) for 48 hours, separated by SDS-PAGE and immunoblotted for clathrin heavy chain (CHC) or actin. (C) Stably-transfected LPA₁/HeLa cells grown in 24-well plates were either mock transfected (siControl) or transfected with clathrin siRNA (siClathrin) for 48 hours prior to treatment with or without 10 µM LPA for 45 minutes. The cells were fixed and processed for whole-cell ELISA to quantify surface LPA₁ receptors as described in Materials and Methods. LPA₁ internalization is expressed as the percentage difference in surface LPA₁ between unstimulated cells and agonist-stimulated cells. The data are the mean±s.e.m. of six replicates/siRNA sample combined from two independent experiments. ^**P<0.01 compared to levels in the siControl. (D) Stably transfected LPA₁/HeLa cells were treated with clathrin siRNA for 48 hours prior to incubation with FITC-labeled mouse anti-CD59 and Alexa 594-Tfn for 30 minutes and fluorescence visualization of anti-CD59 and Alexa 594-Tfn labeling. Bar, 10 µm.

Return to article