Journal: Frontiers in Microbiology

Article Title: Candida albicans : The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length

doi: 10.3389/fmicb.2017.01235

Figure Lengend Snippet: C. albicans SC5314 strain induced host cell biphasic ERK 1/2 activation and cortactin phosphorylation by SFKs. HeLa cells were incubated with blastospores from the SC5314 strain for the indicated time points. After incubation, HeLa cells were harvested, lysed, and their protein lysates separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot using the indicated antibodies. The SC5314 strain induced (A) a biphasic activation of host ERK 1/2 (the other isolates did not, not shown) and also (B) the phosphorylation of cortactin (pCort [Y466]) by SFKs. These are representative observations from at least three independent experiments. SC5314, 997,5 g, oral L3837 and blood L3881 isolates induced host cell signaling activation. HeLa cells were incubated with blastospores from four C. albicans isolates for the indicated time points. After incubation, HeLa cells were harvested, lysed, and SFK and cortactin activation were analyzed by Western blot using the indicated antibodies. Cortactin, SFKs and actin have different molecular weights (80–85, 60, and 45 kDa, respectively), thus the nitrocellulose membrane was cut into three horizontal strips, blocked and each strip was incubated with anti-pY466 cortactin, anti-pY416 SFK or anti-actin. (C) pSFK (Y416) and (D) pCort (Y466), cortactin phosphorylated by Src at the Y 466 residue. Actin was used for protein loading normalization. Actin labeling is duplicated in (C,D) . pSFK/Act = densitometric rate of pSFK over actin. pCort/Act = densitometric rate of pCort over actin. These are representative observations from at least three independent experiments.

Article Snippet: Rabbit anti-pT202/Y204 ERK1/2 (#9101S) and rabbit anti-pY416 SFK (#2101S) were obtained from Cell Signaling, Beverly, MA, USA.

Techniques: Activation Assay, Phospho-proteomics, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Membrane, Stripping Membranes, Residue, Labeling

Journal: The Journal of Cell Biology

Article Title: Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex

doi: 10.1083/jcb.201408103

Figure Lengend Snippet: VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm 2 laminar shear for 1 min. Activation of SFKs (SFK pY416 , ∼55 kD) and VEGFR2 (VEGFR2 pY1175 , ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad −/− cells reconstituted with VEcad WT , Ncad VE-TMD , Ncad WT , and VEcad N-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85 pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.

Article Snippet: Other phospho-antibodies used for immunoblotting include rabbit anti-SFK pY416 (#6943; Cell Signaling Technology), rabbit anti-p85 pY458 (#4228; Cell Signaling Technology), rabbit anti-Akt pS473 (700392; Invitrogen), and rabbit ant-p65 pS536 (#3033; Cell Signaling Technology).

Techniques: Infection, shRNA, Shear, Activation Assay, Western Blot, Control, Immunoprecipitation, Phospho-proteomics

Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h and then lysed. Equal amounts of the whole cell lysates (WCLs) were incubated with anti-lamin A/C or pre-immune serum (IgG) as the control. The immunonocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-lamin A/C antibodies. An equal amount of WCLs was analyzed by immunoblotting with anti-Src, anti-Src pY416, or anti-actin. The tyrosine phosphorylation of lamin A was quantified and expressed as -fold relative to the level of MCF7 without dasatinib. (B) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h. The cells were fixed and stained for lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The nuclear circularity (4π × area/perimeter 2 ) was determined. The P -values were calculated from at least 150 cells pooled from three independent experiments. The percentage of the cells with nuclear lobulation was measured (n ≥ 400). The values (mean ± SD) are from three experiments. *** P < 0.001. (C) MDA-MB-231 cells were infected with lentivirues capable of expressing FLAG-lamin A or the mutants (Y45F and Y45D) and selected in the medium with neomycin (neo). An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (D) The cells as described in panel C were fixed and stained for FLAG-lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The percentage of the cells with nuclear lobulation was measured (n ≥ 900). Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available for this figure.

Article Snippet: The rabbit polyclonal anti-Src pY416 (MAB2685) antibody was purchased from R&D Systems.

Techniques: Incubation, Control, Western Blot, Phospho-proteomics, Staining, Infection, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Pattern of Enhancement of Src Kinase Activity on Platelet-derived Growth Factor Stimulation of Glioblastoma Cells Is Affected by the Integrin Engaged

doi: 10.1074/jbc.m304685200

Figure Lengend Snippet: FIG. 9. PDGF stimulation results in Fyn activation in U-87MG cells adherent to laminin. A and B, serum-starved U-87MG cells were harvested with buffered EDTA, resuspended in serum-free media and plated onto laminin (LM) or collagen (COL)-coated plates (5 h), and then stimulated with 83 pM PDGF or vehicle (10 min, 37 °C, 5% CO2), lysed, 300 g of lysate immunoprecipitated with the antibody indicated, the immunoprecipitates subjected to SDS-PAGE, transferred to Immo- bilon and blotted with anti-Src[pY416] IgG (A), stripped, and reprobed with anti-Lyn IgG or anti-Fyn IgG (B). C and D, U-87MG cells were harvested and resuspended as in Fig. 1, incubated with 20 g/ml neutralizing anti-integrin antibody (20 min, 22 °C), and then plated onto a 96-well plate previously coated with collagen or laminin and allowed to attach for 60 min (37 °C, 5% CO2). Wells were washed two times with PBS, the cells harvested with trypsin and counted in a scintillation counter. Attachment to ovalbumin was subtracted. Condi- tions were assayed in replicas of five and the data analyzed and pre- sented as the mean S.E. The experiment was repeated two times.

Article Snippet: The following rabbit antibodies were purchased: anti-phosphospecific Src [pY416] IgG (Santa Cruz Biotechnology, Inc.) (detects only activated Src), anti-phosphospecific Src[pY418] IgG (BioSource International) (detects only activated Src), anti-FAK (focal adhesion kinase) IgG (Upstate Biotechnology Inc.), anti-phosphospecific FAK(pY397) IgG (BioSource International), anti-PDGFr IgG (Santa Cruz Biotechnology, Inc.), anti-PDGFr IgG (Santa Cruz Biotechnology, Inc.), and anti-PDGFr IgG that recognizes both receptor forms (Upstate Biotechnology Inc.).

Techniques: Activation Assay, Immunoprecipitation, SDS Page, Incubation

Journal: The Journal of Cell Biology

Article Title: Quantitative proteomics identifies a Dab2/integrin module regulating cell migration

doi: 10.1083/jcb.200812160

Figure Lengend Snippet: Steady-state surface levels of integrins α1 and β1 but not αv increase in Dab2-deficient cells. (A) Fixed, nonpermeabilized control and Dab2-deficient HeLa cells were analyzed by FACS. (B) Mean and standard error of fluorescent intensity from four independent experiments are shown. (C) FACS analysis of fixed, permeabilized HeLa cells shows that total integrin β1 only increases slightly on Dab2 removal. RT-PCR for integrin β1 and actin mRNA shows equal mRNA levels in control and Dab2-deficient cells. Mean and standard error from five independent experiments are shown. (D) HFFs treated with control or Dab2 siRNA. Western blot showing decreased Dab2 protein and FACS results showing increased surface integrin β1 (mean and standard error of two independent experiments). Data for total integrin β1 level in Dab2-deficient HFFs are shown in Fig. S1 B . (B and D) Dashed lines indicate the control levels. #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test. 2° Ab, secondary antibody control; con, control; ERK, extracellular signal-regulated kinase.

Article Snippet: Other antibodies included mouse anti–integrin α1 (TS2/7; Santa Cruz Biotechnology, Inc.), rabbit anti-Dab2 (Santa Cruz Biotechnology, Inc.), mouse anti-Dab2 (BD), mouse anticlathrin (X22; Abcam), mouse antiadaptin (clone AP.6; EMD), mouse anti-T7 (EMD), mouse anti-TfnR (Ab-1; Abcam), rabbit anti–phospho FAK (pY 576 ; Invitrogen), rabbit anti-FAK (Santa Cruz Biotechnology, Inc.), rabbit anti–phospho Src (pY 416 ; Cell Signaling Technology), rabbit anti-Numb (C29G11; Cell Signaling Technology), rabbit anti-ARH (gift from L. Traub, University of Pittsburgh, Pittsburgh, PA), rabbit anti-Fyn (FYN3; Santa Cruz Biotechnology, Inc.), mouse antivinculin (hVIN-1; Sigma-Aldrich), mouse anti-EEA1 (BD), rabbit anti-EEA1 (Thermo Fisher Scientific), and mouse anti–extracellular signal-regulated kinase (BD).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of Cell Biology

Article Title: Quantitative proteomics identifies a Dab2/integrin module regulating cell migration

doi: 10.1083/jcb.200812160

Figure Lengend Snippet: Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in . (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test.

Article Snippet: Other antibodies included mouse anti–integrin α1 (TS2/7; Santa Cruz Biotechnology, Inc.), rabbit anti-Dab2 (Santa Cruz Biotechnology, Inc.), mouse anti-Dab2 (BD), mouse anticlathrin (X22; Abcam), mouse antiadaptin (clone AP.6; EMD), mouse anti-T7 (EMD), mouse anti-TfnR (Ab-1; Abcam), rabbit anti–phospho FAK (pY 576 ; Invitrogen), rabbit anti-FAK (Santa Cruz Biotechnology, Inc.), rabbit anti–phospho Src (pY 416 ; Cell Signaling Technology), rabbit anti-Numb (C29G11; Cell Signaling Technology), rabbit anti-ARH (gift from L. Traub, University of Pittsburgh, Pittsburgh, PA), rabbit anti-Fyn (FYN3; Santa Cruz Biotechnology, Inc.), mouse antivinculin (hVIN-1; Sigma-Aldrich), mouse anti-EEA1 (BD), rabbit anti-EEA1 (Thermo Fisher Scientific), and mouse anti–extracellular signal-regulated kinase (BD).

Techniques: Western Blot, Transfection