Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: EGF induces Dab1 phosphorylation. Western blot analysis of protein extracts from HEK293 ( A ) and NIH3T3 ( C ) cells expressing EGFR and Dab1. Cells were either left untreated (Lane 1) or treated with human EGF (10 ng/mL) for 3 (Lane 2), 10 (Lane 3), or 20 min (Lane 4). Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Dab1 phosphorylation levels (Panel A1/C1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blots were stripped and re-probed for Dab1 using Ab D4 (panel A2/C2). Extracts were blotted with an antibody specific for phosphorylated EGFR (Tyr 1173) (panel A3/C3). As a loading control, GAPDH was used (Panel A4/C4). Anti-phosphotyrosine signal was normalized to the anti-Dab1 signal and relative levels of phosphorylated Dab1 from 4 independent experiments in HEK293 ( B ) and NIH3T3 ( D ) are presented. The levels of Dab1 phosphorylation in the untreated controls were set to 1. Data were analyzed by an unpaired, two-tailed t -test. ( E ) Primary neuronal cultures (DIV7) were treated with Reelin conditioned medium (RCM, Lane 2), Mock conditioned medium (MCM, Lane 1) for 20 min or with 20 ng/mL murine EGF for 3 min (Lane 4) or left untreated (Lane 3). Cells were lysed and the protein extracts were subjected to immunoprecipitation using an antibody specific to Dab1 (Ab 54). Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blot was stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for phosphorylated EGFR (Tyr 1173) (Panel 3), total levels of EGFR (Panel 4). As a neuronal loading control, Ab NeuN was used (Panel 5). The extracts and precipitates were analyzed by western blotting. Bands were scanned with ChemiDoc Touch Imaging System (BioRad), the anti-phosphotyrosine signal was normalized to the anti-Dab1 band and relative levels of phosphorylated Dab1 from 3–4 independent experiments are presented. The levels of Dab1 phosphorylation in the untreated or mock controls were set to 1. Relative intensity of the presented blot is shown in a box above the first panel. Data were analyzed by way of an unpaired, two-tailed t -test, * p ≤ 0.05, ** p ≤ 0.01, ns, not significant; dotes, number of experiments. Error bars represent standard deviation.

Article Snippet: Afterwards, the dishes were supplemented with 10 ng/mL of human EGF (PHG0315, Thermo Scientific, Waltham, MA, USA) or 20 ng/mL of murine EGF (130-094-036, Miltenyi Biotec, Bergisch Gladbach, Germany) and were incubated for 3, 10, or 20 min at 37 °C + 5% CO 2 .

Techniques: Phospho-proteomics, Western Blot, Expressing, Immunoprecipitation, Control, Two Tailed Test, Imaging, Standard Deviation

Journal: Journal of visualized experiments : JoVE

Article Title: Dissection and isolation of murine glia from multiple central nervous system regions

doi: 10.3791/61345

Figure Lengend Snippet: Table of Materials

Article Snippet: Name of Material/ Equipment Company 0.05% Trypsin and 0.53 mM EDTA Gibco 12-Well Plates Greiner Bio-One 1X PBS pH 7.4 Gibco 32% Paraformaldehyde Electron Microscopy Sciences 50 mL, 25 cm 2 cell culture flask Greiner Bio-One Antibiotic-Antimycotic 100X Gibco B-27 Supplement 50X Gibco Bovine serum albumin Sigma Confocal Microscope Zeiss DAPI ThermoFisher DMEM (1X), high glucose with Na pyruvate Gibco Dnase I Sigma Fetal bovine serum heat inactivated Gibco Fibronectin from bovine plasma Sigma Fine stitch Scissors Sklar Goat anti-rabbit IgG Alexa Fluor 488 Invitrogen Goat anti-rat IgG Alexa Fluor 555 Invitrogen Hanks' Balanced Salt Solution (w/o Ca or Mg) ThermoFisher L-glutamine, 200mM Gibco Murine epidermal growth factor ThermoFisher Murine IFN-γ Peprotech Murine PDGF-AA Peprotech Neurobasal Gibco Normal goat serum Sigma Operating Scissors Surgi-OR Poly-D-Lysine 12 mm #1 German Glass Coverslip Corning Biocoatt Prolong Gold Antifade Reagent Cell Signaling Technology Rabbit anti-Iba1 Wako Rabbit anti-NG2 Chondroitin Proteoglycan Millipore Rat anti-GFAP ThermoFisher Rat anti-myelin basic protein Abcam Sharp Tip Scissors Surgi-OR Stereo Microscope Leica Tissue Forceps Sklar Triton X-100 Fisher Bioreagents Trypsin Inhibitor (from chicken egg white) Sigma Catalog Number Comments/Description 25300054 Tissue dissociation 665 180 Cell culture plate 10010031 Standard reagent 15714-S Fixative 690 175 Cell culture (T25) flask 15240-096 Media component 17504-044 Media component A9647-50G Antibody diluent LSM 800 Confocal for imaging D1306 Nuclear stain 11995040 Media component 10104159001 Tissue dissociation A3840001 Media component F1141-1MG Cell adherent 64-3260 Dissection tools A11008 Secondary staining antibody A21434 Secondary staining antibody 14170120 Tissue dissociation 20530081 Media component PMG8044 Media component 315-05-20UG Media component 315-17 Media component 21103-049 Media component G9023 Blocking solution component 95-272 Dissection tools 354086 Cell adherent 9071S Mounting Media 019-19741 Primary antibody ab5320 Primary antibody 13-0300 Primary antibody ab7349 Primary antibody 95-104 Dissection tools S4 E Stereo Zoom Microscope Microscope for dissection 66-7644 Dissection tools BP151-100 Cell permabilization 10109878001 Tissue dissociation Open in a separate window Table of Materials.

Techniques: Electron Microscopy, Cell Culture, Microscopy, Clinical Proteomics, Imaging, Staining, Dissection, Blocking Assay