Journal: Immunity

Article Title: Fever Promotes T Lymphocyte Trafficking via a Thermal Sensory Pathway Involving Heat Shock Protein 90 and α4 Integrins

doi: 10.1016/j.immuni.2018.11.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hamster monoclonal FITC anti-β1 integrin , Santa Cruz , Cat# sc-19656; RRID: AB_627005.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Cell Isolation, shRNA, Software

Journal: Open Biology

Article Title: Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

doi: 10.1098/rsob.210166

Figure Lengend Snippet: EGFR activity is required for keratinocyte adhesion. ( a ) NHKs stably expressing GFP, siRNA-resistant EGFR-GFP (siR-EGFR-GFP) were transfected with control siRNA (siCtrl) or siRNA targeting EGFR (siEGFR) before plating onto laminin. Cells were left to adhere for 40 min before fixation and immunostaining with vinculin (magenta) and F-actin (cyan). Scale bar 10 µm. Phalloidin was used to quantify the average area per cell. N = 3 * p < 0.05, ** p < 0.01; ( b ) NHKs were pre-treated with DMSO or EGFR inhibitor (AG1478) for an hour before re-plating onto laminin in the presence of AG1478. After 40 min, cells were fixed and immunostained with FAK (magenta), EGFR (green) and F-actin (cyan). Phalloidin was used to quantify the average area per cell. N = 4 * p < 0.05, ** p < 0.01; ( c ) NHKs were plated onto laminin before lysates were collected at 0, 20, 40 and 60 min after matrix adhesion. Western blots were probed for phospho-FAK (Y397), FAK, phospho-EGFR (Y1173) and GAPDH. ( d ) Confluent monolayers of NHKs were pre-treated with AG1478 for an hour before wounding. After an additional hour of inhibitor incubation, immunoprecipitation was carried out using an antibody against FAK. Western blot analysis was performed probed with EGFR and FAK. ( e ) Confluent monolayers of NHKs stably expressing eGFP or EGFR-eGFP were pre-treated with AG1478 (AG), PF228 (PF) or DMSO as a negative control. After 1 h, monolayers were wounded and were incubated for an hour with AG1478/PF228 before cell lysates were collected for immunoprecipitation with GFP-TRAP beads. Western blot analysis was carried out probed with FAK, β1 integrin and GFP.

Article Snippet: Primary antibodies for western blotting were mouse monoclonal anti-GAPDH (Chemicom, Mississauga, ON, Canada), anti-GFP (MBL), anti-HSC70 (Sigma Aldrich, St Louis, MO, USA), anti-Src (Millipore, Burlington, MA, USA) and rabbit polyclonal anti-β1 integrin (Millipore, Burlington, MA, USA), anti-EGFR (Cell Signaling, Danvers, MA, USA), anti-Erk1/2 (Cell Signaling, Danvers, MA, USA), anti-ezrin (Biotechne, Minneapolis, MN, USA), anti-FAK (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-EGFR (Y1068, Cell Signaling, Danvers, MA, USA), anti-phospho-EGFR (Y1137, Cell Signaling, Danvers, MA, USA), anti-phospho-Erk1/2 (T202, Y204, Cell Signaling, Danvers, MA, USA), anti-phospho-ezrin (Y478, Abcam, Cambridge, UK; noting the authors performed validation experiments to demonstrate the specificity of this antibody to ezrin; not shown), anti-phospho-FAK (Y397, Cell Signaling, Danvers, MA, USA) and anti-phospho-Src (Y418, Millipore, Burlington, MA, USA).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunostaining, Western Blot, Incubation, Immunoprecipitation, Negative Control