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: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: NRG1 treatment increases ERBB4 activation in gastrocnemius muscle. C57BL/6JRJ (C57) control and db/db (Db) male mice were treated with vehicle (VHL; 0.9% NaCl solution; n = 8/each condition), or with NRG1 (50 μg . kg −1 ; n = 8/each condition), three days per week for eight weeks. Western blot analysis ( A ) cropped images) was used to quantify in gastrocnemius muscle samples the abundance of full length (115 kDa) ( B ) and cleaved (42 kDa) ( C ) NRG1 and the NRG1 cleavage index (the ratio between cleaved and full length NRG1) ( D ) as well as the abundance ( E ) and phosphorylation ratios ( F ) of ERBB2, ERBB3 and ERBB4. Results are the mean ± SEM (n = 8 per group) relative to the level in untreated healthy mice (C57-VHL, white bars). Diabetes (healthy vs db/db mice) and NRG1 (saline vs NRG1) effects were investigated with a 2 × 2 ANOVA. When a significant interaction was found, the Tuckey’s test was used for post-hoc multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: Summary of the main effects of diabetes and NRG1 treatment in gastrocnemius muscle. The abundance of ERBB receptors is increased in diabetic db/db mice. This is associated with increased phosphorylation ratio of ERBB2 and decreased phosphorylation ratios of ERBB3 and ERBB4. Similarly, phosphorylation (activation) of the metabolic regulators AMPK, ACC and ACL is reduced in diabetic mice as well as Pparb and Tfam mRNA expression level. However, mitochondrial respiration in permeabilised fibres is similar in diabetic and control healthy mice. Chronic NRG1 treatment increases ERBB4 phosphorylation and tends to decrease ERBB3 phosphorylation. Among the regulators of the mitochondrial biogenesis pathway, only Pparb mRNA expression is slightly increased by NRG1. However, in NRG1-treated mice, complex 2-mediated mitochondrial respiration and complex 2 subunit abundance are increased. Effect of diabetes: red arrows. Effect of NRG1: green arrows. Figure was produced using Servier Medical Art image bank ( http://www.servier.com/Powerpoint-image-bank ), which is licensed under a Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Produced

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor as a Biologic Switch in Hair Growth Cycle

doi: 10.1074/jbc.m212082200

Figure Lengend Snippet: FIG. 4. Immunoblots of crude skin samples from specific time points. a, on both Day 7 and Day 14, expression and phosphorylation of EGFR were detected in both wild type and transgenic mice. On Day 21 and Day 35, normally the telogen phase, both the expression level and phosphorylation level of EGFR were intensively down-regulated. How- ever, samples from transgenic animals had comparatively higher levels of EGFR expression and phosphorylation than samples from wild type animals. Complete down-regulation of ErbB2 and ErbB3 expression was observed on Day 35 in wild type but not in transgenic skin. Com- parable amounts of the mature form of EGF were detected at each stage in transgenic and wild type samples. b, immunoprecipitation of ErbB2 and ErbB3. On Day 14, no observable difference between wild type and transgenic skin was detected with respect to phosphorylation of ErbB2 and EGFR/ErbB2 heterodimerization. Similar results were obtained for ErbB3. On Day 35 (telogen), only transgenic skin showed EGFR/ErbB2 heterodimerization and phosphorylation.

Article Snippet: The primary antibodies and working concentrations are as follows: rabbit anti-mouse EGF (Upstate Biotechnology, Inc., 2 g/ml); mouse anti-human EGFR (Transduction Laboratories, 1 g/ml); rabbit anti-mouse ErbB2 (Santa Cruz Biotechnology, 0.2 g/ml); rabbit anti-mouse ErbB3 (Santa Cruz Biotechnology, 0.2 g/ml); and mouse anti-phosphotyrosine PY20 (Transduction Laboratories, 1 g/ml).

Techniques: Western Blot, Expressing, Phospho-proteomics, Transgenic Assay, Immunoprecipitation