EGFR Surface Expression Assay

Electronic supplementary material

SupplementaryMethods

EGFR surface expression assay

Oocytes expressing wild-type or mutant EGFR were blocked for 1-2 hours at 4°C in OR2(+) medium (5mM KCl, 5mM NaOH, 1mM MgCl2, 1.8mM CaCl2, 82.5mM NaCl, 5mM HEPES) containing 1% bovine serum albumin (BSA)), labeled overnight at 4°C with a rat monoclonal antibody raised against the extracellular domain of human EGFR (3H2090, Santa Cruz Biotechnology, INC, 1 μg/ml in OR2(+)/BSA solution) and extensive washed (OR2(+)/BSA solution, 2hours. 4°C). Following incubation with an HRP-coupled goat anti-rat secondary antibody (KPL, cat# 474-1612, 2 μg/ml in OR2(+)/BSA, 1-1.5h at 4°C) cells were washed (OR2(+)/BSA, 1-1.5h at 4°C) and transferred to OR2(+) without BSA. Individual oocytes were placed in 100 ul of SuperSignal Elisa Pico chemiluminescent substrate (Thermo Scientific, cat#37070) and incubated at room temperature for 1 min. Chemiluminescence was quantified in a TD-20E luminometer (Turner Designs, CA). Chemiluminescence intensity for each oocyte was expressed as a percentage of the mean chemiluminescence intensity obtained for the wild-type EGFR expressing oocytes in the same experiment. Surface labeling for non-injected oocytes was les than 0.5%.

Extraction of acidic lipids and ELISA PIP2 assay.

For extraction of acidic lipids, membranes isolated from wortmanin-treated (15μM, 1h, R.T.) and control oocytes were solubilized in oocyte lysis buffer (5mM Tris-HCl pH: 8.0, 1mM EDTA, 1mM EGTA containing protease and phosphatase inhibitors) and TCA precipitated (0.5M TCA, 10min, 4oC). After centrifugation (2000rpm, 5min 4oC) the pellet was washed 2 times with 5%TCA/1mM EDTA and re-suspended with MeOH:CHCl3 (2:1) for extraction of neutral lipids (10 min, RT). Following centrifugation (2000rpm, 5min R.T.) and removal of neutral lipids, acidic lipids were extracted in MeOH:CHCl3:12M HCl (80:40:1) (15 min RT), phases were split by mixing with an equal volume of CHCl3:0.1M HCl (1:2) and centrifugation (2000rpm, 5min R.T.) and the lower phase was collected, dried and stored at -20oC.

PIP2 levels were quantified using a PIP2 Mass ELISA kit (K-4500, Echelon Biosciences Inc, Salt lake City, UT) according to manufacturer’s directions. A standard curve generated usingknown amounts of PIP2(Sigmoidal dose response fit, Microcal Origin 6 software) was used for determination of PIP2 levels in our samples. PIP2 levels in each of the wormanin-treated samples were normalized for PIP2levels in the control non-treated sample from the same experiment and data were expressed as mean ± S.E.

FACS Analysis

48h after siRNA transfection Hela cells were detached from the plate to the point of single cell suspension using Cellgro cell-stripper solution, collected with 2% FBS in PBS, spun, washed with 2% FBS in PBS and blocked in 2% FBS in PBS at 4 oC for 30 min. A monoclonal primary antibody against an EGFR extracellular epitope (255, Oncogene Research Products, 5µg/ml) was added in and incubated at 4oC for 2 h. The cells were then washed with 2% FBS in PBS, resuspended in 2% FBS in PBS and FITC-labeled secondary antibody (goat anti-mouse, Jackson Laboratories, 1:1000) was added at 4oC for 1h. Cells were then washed twice with 2% FBS in PBS and resuspended in 600 ml of 2% FBS in PBS and placed in tubes for FACS analysis.

Legends forSupplementary Figures

Figure S1. Mutations in the JD of EGFR that impair its phosphorylation do not interfere with the surface expression of the receptor. Data summary (mean ± S.E.) of relative (% of wild-type) surface expression levels for wild-type and mutant EGFR from 2 independent experiments (number of oocytes analyzedin each experiment is indicated on the top of each bar).Total EGFR levels in the membrane fraction of these oocytes were similar to those presented in Figure 2B.

Figure S2. Wortmannin treatment reduces PIP2 levels. Oocytes were incubated for 1h in the presence and absence of wortmannin (15µM) and subsequently lysed for preparation of the membrane fraction. Extraction of membrane lipids and PIP2 ELISA assay are described under Supplemental Methods. Data points corresponding to known amounts of PIP2 were fitted to the equation y = A2 + (A1-A2)/(1 + (x/x0)p) with

A1=2.14965±0.42526, A2=-0.05576±0.75398, x0=20.3308524.0873 and p=0.52726±0.39675. PIP2 levels in each sample,calculated from the standard curve(left panel),werenormalized for PIP2 levels in the control non-treated sample from the same experiment.Data from 3 experiments arepresented as mean ± S.E (right panel). Statistical significance is indicated.

Figure S3.Downregulation of PI(5)PKI does not affect cell surface expression of EGFR a) Histograms of mean EGFR extracellular fluorescence intensity measured by FACS analysis, as described in SupplementaryMethods. Background was estimated by performing the experiment in the absence of primary antibody (Negative, neg). The rest of the experimental conditions correspond to Fig. 4c. b) Bars representing the effect of PIPKI siRNA transfection on surface EGFR in the presence and absence of EGF (%±SE) were calculated from three experiments for each condition. The means were not significantly different in the presence of siRNA (t-test).