Supplemental Material

This article contains the following supporting material:

• Supplementary Figure S1 - N-ERMAD(E244K) changes the distribution of endogenous ezrin and the reorganisation of F-actin in response to protein kinase C activation. MDA-MB-231 cells expressing N-ERMAD(E244K)-mRFP after microinjection, and the organisation of F-actin and ezrin was visualised by Alexa Fluor-488 phalloidin and mAb 2H3, respectively, and followed by secondary Cy5-conjugated anti-mouse, untreated (A) or treated 20 min with 1µM TPA (B). (C) Quantification of F-actin in un-transfected and N-ERMAD(E244K) expressing MDA-MB-231 cells, untreated or treated with 1µM TPA. Fluorescent intensity of Alexa-488 conjugated phalloidin was quantified using ImageJ. Bars indicate average ± SEM, n=10 cells.
• Supplementary Figure S2 - Raft proteins were obtained by buoyant-density fractionation over a discontinuous OptiPrep density gradient.2x10⁷ cells were washed in cold PBS and lysed in 400 μl OptiPrep lysate buffer for 30 min at 4ºC. The lysate was clarified by centrifugation at 250g for 5 min. The post-nuclear supernatant was then adjusted to 40% iodixanol (by adding 800μl OptiPrep solution), overlaid on other layers of 0.6 ml of 35%, 30%, 25%, and 20% iodixanol and then topped with a layer of 0.6 ml lysate buffer. Buoyant-density centrifugation was performed at 35,000 rpm for 18 h at 4ºC in an ultracentrifuge. 0.3ml fractions were collected from the gradient. Proteins were precipitated from these fractions in 6.5% TCA overnight, washed with cold 80% acetone, then denatured in Laemmli's sample buffer, separated by 1-D SDSPAGE.