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: Immunity

Article Title: The Strength of T Cell Receptor Signal Controls the Polarization of Cytotoxic Machinery to the Immunological Synapse

doi: 10.1016/j.immuni.2009.08.024

Figure Lengend Snippet: cSMAC Activation Correlates with Avidity (A and B) Target EL4 cells pulsed with (A) 1 μM OVA 257-264 or (B) G4 peptides, conjugated to in vitro activated OT-I CTLs, stained with anti-Lck (488, green) and pY416 (546, red). Images are shown as projections through the xy plane, or xz plane reconstructed from a 1 μM section across the synapse (en face). Scale bars represent 1.5 μM. (C) Graph showing the percent maximum tetramer stained OT-I relative to time zero for OVA 257-264 -K b -PE, and G4-K b -PE tetramers over 1 hr at 37°C, detected by flow cytometry. (D and E) Conjugates prepared as in (A) and (B), but preincubated with orthovanadate. (F) The mean percent (from two experiments) of Lck + staining synapses with distinct pY416 accumulation in the presence or absence of sodium orthovanadate.

Article Snippet: For the immunofluorescence studies, reagents were obtained from the following: mouse anti-actin (AC-40), rabbit anti-actin, and rabbit anti-γ-tubulin (Sigma-Aldrich); mouse anti-mouse Lck (3A5) (Millipore); rat anti-mouse CD8 (YTS192) (H. Waldmann, Oxford University); rat anti-mouse CD107a (LAMP-1, 1BD4) (Developmental Studies Hybridoma Bank, IA); mouse-anti-Phospho-ERK (pT202/pY204) (BD Biosciences); rabbit polyclonal anti-mouse-phospho-Src-family (pY416) (Cell Signaling); and Hoechst 33342 (Invitrogen).

Techniques: Activation Assay, In Vitro, Staining, Flow Cytometry