Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet:

Article Snippet: Primary antibodies anti-Mfn2 (Cell Signaling 9482S), anti-Mfn1 (Abcam, ab126575), anti-pan-CaMKII (Cell Signaling #3362), anti-phosphor-CaMKII (Thr286) (Cell Signaling #12716), anti-phospho myosin light chain 2 (Cell Signaling #3671), anti-myosin light chain 2 (Cell Signaling #3672), anti-phospho-PAK (Cell Signaling #2605S), anti-PAK (Cell Signaling #2604), and secondary antibody HRP AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, #111-035-003), goat anti-mouse IgG Alexa Fluor 680 (Invitrogen, #A28183), and goat anti-rabbit IgG Alexa Fluor Plus 800 (Invitrogen, #A32735).

Techniques: Transfection, Construct, Over Expression, Expressing, Dominant Negative Mutation, Recombinant, Plasmid Preparation, Control, Knockdown, Sequencing, Staining, Activation Assay, Imaging, Cloning, Software, Microscopy

Journal:

Article Title: Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways

doi:

Figure Lengend Snippet: Expression of Tpr-Met protects cells from apoptosis in an Akt-dependent manner. (A) Akt1 and Akt2 are activated by Tpr-Met in NIH 3T3 cells. (Upper) Akt kinase activity. (Lower) Western blot analysis of immunoprecipitates by using anti-HA antibody, demonstrating equivalent loading in each lane. (B) Untransfected cells and cells transiently transfected with Tpr-Met or Tpr-Met plus dominant-negative mutants Akt1AA and Akt2E299K were serum starved and UV irradiated. Cells were then fixed and stained with anti-Met antibody followed by FITC-conjugated goat anti-rabbit IgG. The frequency of apoptotic cells was assessed by Hoechst 33342 staining and expressed as percent of total cell number. Bar = mean ± SD of three independent experiments. Cells expressing Tpr-Met were resistant to apoptosis induced by serum starvation and UV irradiation; dominant-negative forms of Akt1 and Akt2 blocked the anti-apoptotic effect of Tpr-Met. (C) Representative staining of transfectants of Tpr-Met (Left) and Tpr-Met plus Akt1AA and Akt2E299K (Right). (Upper) Green fluorescence of anti-Met staining. (Lower) blue fluorescence of Hoechst 33342 staining of apoptotic and nonapoptotic cells of the same field. Arrows indicate nuclei of Tpr-Met-positive cells shown in corresponding Upper panels.

Article Snippet: Expression of Tpr-Met was examined by staining with rabbit anti-human Met antibody (Santa Cruz Biotechnology), which shows no crossreaction with endogenous Met of murine NIH 3T3 cells (data not shown), followed by FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch).

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Dominant Negative Mutation, Irradiation, Staining, Fluorescence

Journal: eLife

Article Title: Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies

doi: 10.7554/eLife.59594

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Article Snippet: Antibody , Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L) , Jackson ImmunoResearch (West Grove, PA, USA) , 111-605-045; RRID: AB_2338075 , IHC (1:500).

Techniques: Sequencing, Dominant Negative Mutation, Gel Extraction, Software, Staining, Membrane, Western Blot

Journal: eLife

Article Title: Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies

doi: 10.7554/eLife.59594

Figure Lengend Snippet:

Article Snippet: Antibody , Cy3 AffiniPure Goat Anti-Rabbit IgG (H+L) , Jackson ImmunoResearch (West Grove, PA, USA) , 111-165-003; RRID: AB_2338000 , IHC (1:500).

Techniques: Sequencing, Dominant Negative Mutation, Gel Extraction, Software, Staining, Membrane, Western Blot

Journal: PLoS Pathogens

Article Title: GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

doi: 10.1371/journal.ppat.1000228

Figure Lengend Snippet: XY, XZ and YZ sections as well as three-dimensional reconstructions (volume render) of confocal image series of GEF-H1 recruitment to the invasion sites of S. flexneri . Polarized MDCK monolayers were exposed to green fluorescent protein (GFP)-expressing S. flexneri and then fixed and stained at different time points. Endogenous GEF-H1 was stained with anti-GEF-H1 antibody and Texas red secondary antibody. Cingulin was stained with anti-cingulin antibody and Cy5 secondary antibody. Black arrows locate the cross section level. Bars indicate 5 µm. (A) Initially, S. flexneri was found attached to TJs, co-localizing with GEF-H1 and cingulin. (B and C) Thereafter, Shigella gained access to the TJs and induced membrane ruffles and pedestals that extended above the TJs. White arrows represent area indicated by white frame in (B). GEF-H1 was recruited from tight junctional complexes into bacterial entry sites, while cingulin was not and remained associated with the TJs. (D) Subsequently, Shigella was found within the cytoplasm of MDCK cells and GEF-H1 and cingulin were removed from the tight junctional area of infected cells. (E) Upon cell invasion, Shigella was found inside the epithelial monolayer either free in the cytoplasm or associated with intracellular vesicles containing GEF-H1 (white arrows). The cytoplasm of the infected cells appeared to retract and the neighboring epithelial cells started to close above the infected cells.

Article Snippet: FITC conjugated anti-rabbit IgG, Texas red conjugated anti-mouse and anti-rabbit IgG and Cy5 conjugated anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA). γTriDAP and αTriDAP were purchased from AnaSpec (San Jose, CA).

Techniques: Expressing, Staining, Membrane, Infection

Journal: PLoS Pathogens

Article Title: GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

doi: 10.1371/journal.ppat.1000228

Figure Lengend Snippet: (A) NF-κB activation in response to active and inactive NOD1 ligands in the absence or presence of GEF-H1 and NOD1 siRNA in HEK293 cells. Bars represent mean±SD (* p<0.01, compared to responses in the presence of control siRNA and γTriDAP) . (B) NF-κB activation in response to overexpression of NOD1 in the presence of control or GEF-H1 siRNA in HEK293 cells. Bars represent mean±SD (* p<0.01 compared to control siRNA) . (C) NF-κB activation in response to transfection of indicated amounts of expression vectors encoding GEF-H1 and NOD1 in HEK293 cells. Bars represent mean±SD (*p<0.01 compared to GEF-H1 and NOD1 alone) . (D and E) RhoA activation is not required for γTriDAP and TNFα signaling. NF-κB activation in response to active and inactive NOD1 ligands and TNFα in HEK293 cells transfected with GEF-H1 (Y395A), dominant negative RhoA (T19N) or control expression vectors. Bars represent mean±SD (*p = NS, control vector vs GEF-H1 (Y395) or RhoA (T19) in the presence of γTriDAP) . (F) Co-immunoprecipitation of GEF-H1 and NOD1 with indicated antibodies in HEK293 cells. (G) Confocal microscopic image analysis of the subcellular localization of NOD1, GEFH1 and E-cadherin. MDCK monolayers and HEK 293 cells were transfected with HA-NOD1 vector alone or in addition to vsv-GEF-H1 and then fixed and stained for confocal microscopic analysis. Endogenous GEF-H1 was stained with anti-GEF-H1 antibody and Texas red secondary antibody. E-cadherin was stained with anti-E-cadherin antibody and Texas red secondary antibody. HA-NOD1 was stained with anti-HA antibody and Cy5, Texas Red or FITC secondary antibody; vsv-GEF-H1 was stained with anti-vsv antibody and Cy5 or FITC secondary antibody. Bars indicate 5 µm.

Article Snippet: FITC conjugated anti-rabbit IgG, Texas red conjugated anti-mouse and anti-rabbit IgG and Cy5 conjugated anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA). γTriDAP and αTriDAP were purchased from AnaSpec (San Jose, CA).

Techniques: Activation Assay, Control, Over Expression, Transfection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Immunoprecipitation, Staining