Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a-d Confocal immunofluorescence imaging of traced surface labelled integrin β1 and ErbB3: MCF7 cells were labelled on ice with an Alexa488-conjugated anti-integrin β1 antibody prior to incubation for 30 minutes at 37°C to allow integrin β1 internalisation, and subsequent cell-fixation and immunolabelling of ErbB3 (red) and counterstained with DAPI (blue). c Histogram of fluorescence intensities along dotted lines indicated in ( b ). d Analysis of colocalization of integrin β1 and ErbB3. The enrichment of integrin β1 in ErbB3-positive intracellular structures (0.5-2 μm diameter) was determined by the formula (a-b)/b where a is the integrin β1 intensity at ErbB3 positive structures, and b the adjacent intensity (background) for each structure. Average intensity projections of all analysed structures are shown on the right-hand side. e Schematic outline of the recycling assays conducted in ( f-k ) in the absence of growth factors: Briefly, the surface-pool of integrin β1 was labelled with an Alexa488-conjugated antibody and allowed to endocytose. Fluorophore label remaining on the cell surface was quenched with an anti-Alexa488 antibody, prior to visualisation of traced integrin β1 re-emerging on the cell surface by live-cell TIRF microscopy. The recycling assays were conducted after prior transfection with non-targeting control siRNA or siRNA targeting indicated proteins. f Represenative TIRF microscopy images of integrin β1 from peripheral areas of MCF10A cells. g Quantifications of recycled integrin β1 performed on indicated number of cells (outside of brackets on the right-hand side of graphs), from three independent experiments and shown as Alexa488 intensity normalized between 0-1, with the control as reference where F norm =((F max -F min )/(F-F min )). h Representative TIRF microscopy images of integrin β1 from prHMEC cells. i Quantifications of recycled integrin β1 in prHMECs performed as described in ( g ). j , k . Quantified recycling of integrin β1, after prior siRNA-mediated depletion of either EGFR ( j ) or ErbB2 ( k ). Data are presented as mean values ± s.e.m. and P-values determined by two-tailed paired student’s t-test. ns=non significant. Scale bar: 10 μm, except figure 1d (scale bar:1 μm)

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Immunofluorescence, Imaging, Incubation, Fluorescence, Microscopy, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a-d Confocal immunofluorescence imaging of surface-labelled integrin β1 (green) or actin (blue/black) on confluent sheets of MCF10A cells at 0h or 1h after labelling, as outlined in ( a ). Note in ( b ) that depletion of ErbB3 abrogates integrin β1 localisation at the leading front. c Enrichment of integrin β1 determined as ((a-b)/b) where a= mean fluorescence intensity (integrin β1) at a defined area of the leading edge ( c ) or cell-cell contact ( d ) and b=mean intensity of adjacent cytoplasm of same area. Data are presented as mean values (>74 cells per data point) ± s.e.m., n=3 independent experiments. e Scratch closure assay of control or ErbB3-depleted MCF10A cells, cultured in serum-containing but growth factor-deprived media in the presence or absence of the EGFR/ErbB2 inhibitor Lapatinib. Wound area highlighted in yellow. f , g Quantification of scratch aperture ( f ) or area under curve, AUC, ( g ) of samples treated as in ( e ). Data are presented as mean values ± s.e.m., n-values indicated in parenthesis. h Quantification of cell proliferation as incorporation of EdU for indicated times in control or ErbB3 siRNA-transfected cells in the presence or absence of 1 μM lapatinib. Data are presented as mean values ± s.e.m., n=3 independent experiments.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Immunofluorescence, Imaging, Fluorescence, Cell Culture, Transfection

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a Confocal imaging of Alexa594-conjucated transferrin chased with unlabelled holo-transferrin for indicated times in MCF7 cells. Note that siRNA-mediated depletion of ErbB3 caused prolonged intracellular retention of transferrin. b Quantification of Alexa594 fluorescence intensity in cells treated as in a (n>17 cells for each data point from three experiments) normalised against the control siRNA treated, 0 hour timepoint of each independent experiment. b , d , f Data are presented as mean values ± s.e.m. P values determined by two-tailed paired student’s t-test. Scale bar: 10 μm. c experimental outline of the VSVG trafficking experiments ( d and e). d , e Western blot analysis of the surface pool of VSV-G-ts45-GFP (pulldown of surface-biotinylated VSV-G-ts45-GFP), after its release from the endoplasmic reticulum (ER) at permissive temperature for indicated times. Note that ErbB3-depletion did not influence secretive trafficking of VSVG from the ER. e quantification of normalised levels of VSV-G-GFP in biotin-pulldowns as determined by immunoblot band intensities (n=3 independent experiments). Data are presented as mean values ± s.e.m. P determined by two-tailed paired student’s t-test. ns=non significant.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Imaging, Fluorescence, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a , b Confocal immunofluorescence imaging of traced internalised integrin β1: The MCF10A cells were transfected with control or ErbB3 siRNA and assay performed in growth factor deprived media. c Quantification of immunofluorescence intensity of internalised integrin β1 traced for indicated times (n>32 cells per data point from 5 independent experiments). d Determination of integrin β1 turnover by pulse-chase metabolic labelling: Control or ErbB3 siRNA-transfected MCF10A cells were pulse-chase labelled with radioactive ( S) methionine and cysteine. Radiolabelled integrin β was visualised by radiography of immunoprecipitates (upper panel). Cell lysates and immunoprecipitates were analysed by immunoblotting. e Quantification of pulse chased 35S-labelled integrin β1, as in ( d ) (n=4 independent experiments). f , g Confocal immunofluorescence imaging of surface-labelled integrin β1 (using an Alexa488-conjugated anti-integrin β1 antibody), prior to (0 hours) or after tracing at 37°C for 1.5 hours. A scratch was inflicted prior to antibody incubation. Note that application of the lysosome inhibitor chloroquine caused accumulation of integrin β1 in intracellular vesicular compartments both in control of ErbB3-depleted cells. h Quantification of integrin β1 fluorescence intensity in cells bordering the migratory front in samples treated as in g , showing that chloroquine restored levels of integrin β1 in ErbB3-depleted cells. Data presented as mean values ± s.e.m., n=19-27 cells per data point from 3 independent experiments. P values determined by two-tailed paired student’s t-test. ns=non-significant.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Immunofluorescence, Imaging, Transfection, Pulse Chase, Metabolic Labelling, Western Blot, Incubation, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a Confocal imaging of ErbB3-mCherry and indicated Rab marker expressed in MCF7 cells, with or without prior treatment with the recycling inhibitor primaquine (PQ). b Analysis of colocalization of ErbB3-mCherry and Rab4 or Rab11. The relative enrichment of ErbB3 at the Rab-positive structures was determined by the formula (a-b)/b where a is the ErbB3-mCherry intensity of the center of Rab4 structures, and b the adjacent volume (background) for each structure. Each data point represents the average of a minimum of 20 structures in one cell. P-values were determined using unpaired, 2-tailed Student’s t-test. c Average projections of all analysed (indicated number) of GFP-Rab4 or GFP-Rab11 positive structures from indicated number of cells (3 independent experiments).

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Imaging, Marker

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after 30 minutes treatment with primaquine (PQ), showing endogenous binding of ErbB3 with GGA3 and Rabaptin5 that increases upon PQ treatment, and the presumed accumulation of recycling endosomes (representative of 3 independent experiments). b Immunoblotting of Arf6 immunoprecipitates or input cell lysates, following control or ErbB3 siRNA transfection and 10 minutes treatment with PQ or vehicle. Note that endogenous co-precipitation of Arf6 with both GGA3 and rabaptin5 is reduced in the absence of ErbB3. c Quantification of GGA3 and Rabaptin5 protein levels (by western blotting) and mRNA levels (by quantitative RT-PCR) in ErbB3 siRNA-transfected MCF10A cells relative to control-transfected cells (n=4 experiments for protein and n=3 for mRNA). Data are presented as mean values ± s.e.m. and P values (one sample student’s t-test). d Structural model highlighting the putative GGA3-binding motif 864-DxxLL-867 in the ErbB3 kinase domain. e Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after ectopic expression of ErbB3 or the ErbB3 LL866/867AA mutant with GGA3 in HEK293T cells. Note that the LL866/867AA mutant ErbB3 fails to co-precipitate with GGA3. f The LL866/867AA mutation compromises the ability of ErbB3 to promote assembly of the Arf6-GGA3-Rabatin5 sorting complex: Immunoblotting of Arf6 immunoprecipitates or input cell lysates, following ectopic expression of Arf6, GGA3 and Rabaptin5 (Rbtn5), with or without ErbB3 or ErbB3-LL866/867AA.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Western Blot, Binding Assay, Transfection, Quantitative RT-PCR, Expressing, Mutagenesis

Journal: Scientific Reports

Article Title: Effects of neuregulin-1 administration on neurogenesis in the adult mouse hippocampus, and characterization of immature neurons along the septotemporal axis

doi: 10.1038/srep30467

Figure Lengend Snippet: Ventral ( A ) (but not dorsal ( B )) dentate gyrus (DG) ErbB3 phosphorylation is increased by 24 h of subcutaneous NRG1. *p < 0.05. FC, fold change; NRG, neuregulin; SAL, saline.

Article Snippet: ErbB3 phosphorylation was assessed using a phospho-ErbB3-specific ELISA kit (RayBiotech).

Techniques: