(A) Whole extracts of Ln229 and T98 glioblastoma cells were immunoblotted with antibodies to DEP-1 or the p85 subunit of phosphoinositide 3-kinase.
(B) Ln229 cells were transfected with siRNA oligonucleotides targeting DEP-1 transcripts. Following 48h of incubation, cells were harvested for immunoblotting analysis.
(C) Ln229 cells were transfected with siRNA oligonucleotides targeting DEP-1 transcripts, then incubated for 48h and plated in 96-well plates under 1% serum. Proliferation was measured after 3 days and compared to day 1. An asterisk indicates p= 0.001.
(D) HeLa cells were transfected with DEP-1-siRNA (10nM; panels d, e, and f), or with control siRNA (panels a, b, and c), then stimulated with EGF (20ng/ml, 10 min). Thereafter, cells were analyzed by immunofluorescence using an antibody to EGFR or to phosphorylated EGFR (pY1173). Images were taken at the same exposure time. Arrowheads indicate vesicles positive for both EGFR and pY1173; arrows mark EGFR-positive, but pY1173 negative vesicles. Enlarged areas show internalized EGFR in vesicles. Scale bar: 10 μm.
(E) Left panel: The number of cells displaying >10 pY1173-positive vesicles are compared in DEP-1-siRNA- and control- treated HeLa cells. Data are expressed as mean±S.D. (bars) of three independent measurements. Right panel: Fluorescence intensities of pY1173-positive vesicles (n~50; 5 cells) were analyzed using NIH ImageJ software. The experiment was repeated twice.

(A) HeLa cells were transiently transfected with vectors encoding HA-tagged WT DEP-1 (a-c), a CS mutant (d-f), or with a DA mutant (g-i). Thereafter, cells were incubated for 32h, serum-starved for 16h, and fixed. Shown are immunofluorescence images obtained with the indicated antibodies. The merged images (c, f and i) were obtained through ImageJ Stack RGB Merge plugin and indicate co-localization (yellow) of EGFR (green) and DEP-1 (red). Arrows demarcate co-localization at cell borders and junctions. One representative experiments is shown (n=3). Scale bar: 10 μm.
(B) A431 cells were incubated for 2hrs at 4°C with a radio-labeled EGF (20 ng/ml), then washed and subjected to covalent cross-linking with BS³ (1mM). The cross-linking reaction was subsequently quenched. Cell lysates were mixed, in the absence or presence of sodium orthovanadate (0.2mM), with glutathione beads bound to purified GST, GST-DEP-1 (WT), the DA or CS mutants, and incubated for 12h at 4°C. After extensive washing, the samples were resolved by gel electrophoresis and autoradiography (top panel). Staining with Ponceau Red (bottom panel) was used to verify equal gel loading.
(C) HeLa cells were transfected with vectors encoding HA-tagged WT DEP-1, or with the CS or DA mutants. Thereafter, cells were incubated for 32h, serum-starved for 16h, and then pre-incubated (as indicated) for 30 min with a selective EGFR kinase inhibitor (AG1478; 10 μg/ml). This was followed by EGF stimulation (20ng/ml; 10 min). DEP-1 was immunoprecipitated (IP) from whole cell lysates using anti-HA-agarose beads, followed by immunoblotting (IB) with the indicated antibodies.

(A) HeLa cells were transfected with the indicated siRNA oligonucleotides, and 32h later they were starved for 16h and un-treated or treated with EGF (20ng/ml; 10 min). Thereafter, surface localized EGFR was quantified by flow cytometry. Numbers represent percents of initial cell surface EGFR.
(B) HeLa cells transiently expressing HA-DEP-1 (or a control vector) were assayed as in A. One representative experiment (n=2) is shown.
(C) HeLa cells were stimulated with a fluorescently labeled EGF (FITC-EGF; 20ng/ml) for 30 min at 22°C, fixed and analyzed by immunofluorescence using an antibody to DEP-1.
(D) HeLa cells were serum-starved for 16h and stimulated with EGF (20ng/ml) for the indicated time intervals. Thereafter, cells were surface labeled with antibodies to EGFR and DEP-1. The remaining surface fraction of each protein was quantified by flow cytometry.
(E) HeLa cells expressing DEP-1-HA were stimulated with EGF (20ng/ml) for the indicated time intervals. EGFR and the ectopically expressed DEP-1 were immunoprecipitated from cell lysates and subjected to immunoblotting.
(F) HeLa cells were treated as in E and stimulated with EGF (20ng/ml; 5 min). EGFR was analyzed by immunoblotting, either directly of after immunoprecipitation. EGFR phosphorylation was detected using an anti-phosphotyrosine antibody.

(A) HeLa cells were serum-starved for 16h, then treated with either a mixture of H₂O₂ (0.2mM) and sodium orthovanadate (1mM; for 15min; HV), Compound 5 (Cpd5; 20mM; for 30 min) or with ethanol (control; 30 min), and then stimulated with EGF (20ng/ml) for the indicated time intervals. Whole cell lysates were blotted with the indicated antibodies.
(B and C) Mixtures of siRNA oligonucleotides were transfected into HeLa cells, which were then incubated for 32h and serum-starved for 16h. The cells were then stimulated with EGF (20ng/ml) for the indicated intervals and lysed. Whole cell lysates were blotted with the indicated antibodies, including an anti-phosphotyrosine antibody (pY99), and antibodies to the active (pERK) and general forms of ERK (gERK).
(D) HeLa cells were transiently transfected with DEP-1 siRNA oligonucleotides or with control siRNA (each at 10nM), incubated for 32h, serum-starved for 16h and stimulated with EGF (20ng/ml) for the indicated time intervals. Whole cell lysates were immunoblotted with the indicated antibodies.
(E) HeLa cells were treated and processed as in D. EGFR phosphorylation was quantified using densitometric analysis and normalized to total EGFR level. One representative experiments (n=3) is shown.

(A) HeLa cells were transfected with a plasmid encoding HA-tagged DEP-1, or with a control expression vector, then incubated for 32h, serum-starved for 16h, and stimulated with EGF (20ng/ml) for the indicated time intervals. Thereafter, cell extracts were analyzed by immunoblotting.
(B) HeLa cells were treated as in A. EGFR phosphorylation was quantified and normalized. One representative experiment (n=3) is shown.
(C) HeLa cells were treated as in A and stimulated with EGF (20ng/ml) for the indicated intervals. Left panel: cells were processed for immunoblotting and densitometry of pERK1/2 levels (normalized to total ERK2 level; gERK). Right panel: cells were lysed, total RNA was prepared and used for reverse transcription. Real-time PCR was carried out with c-FOS primers.
(D) HeLa cells were transfected with plasmids encoding wild type HA-DEP-1 (a-c, h-k), or a catalytically inactive mutant (CS; d-f, l-n), then incubated for 32h, serum-starved for 16h (a-f) and stimulated with EGF (20ng/ml) for 10min (h-n). Cells were then fixed and analyzed by immunofluorescence using an antibody to HA (a, d, h, l) or to phosphorylated EGFR (pY1173; b, e, i, m). Images of phosphorylated EGFR were taken at the same exposure time. Endogenous phosphorylated EGFR appears green and DEP-1 appears red. One representative experiment is shown (n=2). Scale bar: 10 μm.
(E) HeLa cells expressing DEP-1 (WT or CS) were treated as in D. The histogram compares the fractions of cells (±S.D.) displaying vesicular pY1173 in three independent measurements (>100 cells).

(A-C) MCF-7 cells were transfected with vectors encoding EGFR-mRFP, along with GFP-tagged WT DEP-1 (A), the DA mutant (B) or the CS mutant (C). Thereafter, cells were incubated for 32h, serum-starved for 16h, and stimulated with EGF (20ng/ml; 10 min), prior to fixation and fluorescence measurements. Bar: 20 μm.
(D) The mean FRET efficiency between DEP-1-EGFP and EGFR-mRFP was calculated using the following equation in each pixel and averaged per each cell. FRET efficiency = 1 − τda/τcontrol, where τda is the lifetime displayed by cells co-expressing both DEP-1-EGFP and EGFR-mRFP and control is the mean DEP-1-EGFP lifetime measured in the absence of acceptor. Data are means ± SEM of 16–23 cells from three independent experiments. An asterisk refers to the DA mutant and indicates p< 0.05.

(A and B) HeLa cells were transfected with control or DEP-1-HA plasmids, incubated for 32h, serum-starved for 16h and stimulated with EGF (20ng/ml) for either 5min (A) or for the indicated time intervals. Whole cell lysates were immunoblotted with the indicated antibodies.
(C) A model presenting the effect of DEP-1 on EGFR signalling and endocytosis: a complex comprising EGFR and DEP-1 pre-exists at the surface, especially in highly confluent epithelia. On EGF binding and receptor phosphorylation (P), DEP-1 dephosphorylates EGFR, thereby inhibits receptor ubiquitinylation (Ub) by c-CBL, which decelerates the rate of receptor internalization and diminishes MAPK signals generated at the membrane and in endosomes. When DEP-1 is inactive, for example due to loss of heterozygosity (LOH), EGFR is hyperphosphorylated and accordingly it relays strong signals to MAPK, although it gains fast rates of internalization and degradation.