Journal: bioRxiv

Article Title: CK2-dependent phosphorylation of the Brg1 chromatin remodeling enzyme occurs during mitosis

doi: 10.1101/781781

Figure Lengend Snippet: Representative confocal microscopy images from three different mice show the expression of Brg1 (green), CK2 (red), and phosphorylated histone H3 (PHH3, yellow) in somites of E9.5 mouse embryos. The nuclei were stained with DAPI (blue). The white arrow in the upper panel points to the area of enlargement in the lower panels. The white arrows in the lower panel point to mitotic cells.

Article Snippet: The primary antibodies used were: The rabbit anti-CKIIα (2656), rabbit anti-Baf60c (62265), and rabbit anti-phospho-histone H3 (Ser10; D2C8) conjugated to Alexa Fluor 647 were obtained from Cell Signaling Technologies.

Techniques: Confocal Microscopy, Expressing, Staining

Journal: bioRxiv

Article Title: CK2-dependent phosphorylation of the Brg1 chromatin remodeling enzyme occurs during mitosis

doi: 10.1101/781781

Figure Lengend Snippet: Representative confocal microscopy images from three independent biological replicates show the expression of Brg1 (green), CK2 (red), and phosphorylated histone H3 (PHH3, yellow) on proliferating primary myoblasts. The nuclei were stained with DAPI (blue).

Article Snippet: The primary antibodies used were: The rabbit anti-CKIIα (2656), rabbit anti-Baf60c (62265), and rabbit anti-phospho-histone H3 (Ser10; D2C8) conjugated to Alexa Fluor 647 were obtained from Cell Signaling Technologies.

Techniques: Confocal Microscopy, Expressing, Staining

Journal: Journal of Virology

Article Title: Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

doi: 10.1128/JVI.00469-15

Figure Lengend Snippet: Effect of Rho GTPase depletion on VLP production, Gag intracellular localization, and Gag membrane attachment in T cells. (A to E) Effect of Rac1, RhoA, and Cdc42 depletion on VLP production. Jurkat T cells were transfected with p8.2 (expression of Gag, Gag-Pol, and accessory viral proteins) and with the siRNA control or siRNA against Rac1, RhoA, or Cdc42. (A) Immunoblot analysis for detection of the HIV-1 proteins pr55Gag and CAp24 in cell lysates and in VLPs. Tubulin was used as a loading control. (B) Extracellular virus production measured by quantification of immunoblot images, i.e., the ratio between extracellular CAp24 and intracellular Pr55 Gag + CAp24. Bars show mean values and standard deviations resulting from three independent experiments. The statistical significances of differences were calculated by an unpaired t test. **, P value of <0.01; *, P value of <0.05. (C) Cell viability measured by flow cytometry analysis. (D) Quantification of Rho GTPase depletion after siRNA treatment. (E) Percent transfection measured by flow cytometry analysis. Bars show mean values and standard deviations resulting from three independent experiments. (F to J) Effect of Rac1, RhoA, and Cdc42 depletion on Gag intracellular localization. Jurkat T cells were transfected with p8.2 and with the siRNA control (F), siRNA against Rac1 (G), siRNA against RhoA (H), or siRNA against Cdc42 (I). Cells were fixed at 48 h posttransfection, permeabilized, stained for HIV-1 Gag, and analyzed by confocal microscopy. (J) Percentage of cells with each phenotype, calculated for 50 cells. Bars show mean values and standard deviations resulting from three independent experiments. (K and L) Effect of Rac1 or RhoA depletion on Gag cell membrane attachment. Jurkat T cells were microporated with p8.2 and with the siRNA control or siRNA against Rac1 or against RhoA. Cells were then lysed, and the PNS was used for membrane flotation assays. Intracellular proteins of each gradient fraction were loaded onto an SDS-PAGE gel. (K) The viral Gag and Lamp2 proteins were then revealed by immunoblotting, as indicated. (L) Rac1 and RhoA depletion by siRNA knockdown, in the PNS, shown by anti-Rac1 and anti-RhoA immunoblots, respectively.

Article Snippet: Immunoblottings were performed by using the following antibodies: anti-MAp17 and anti-CAp24 (NIH AIDS Reagent Program); HIV-1 p17 (VU47) rabbit and HIV-1 p24Gag monoclonal (24-4) mouse antisera, mouse anti-RhoA (ARH03), mouse anti-Rac1 (ARC03), and mouse anti-Cdc42 (ACD03) antisera (Cytoskeleton); rabbit anti-Wave2 (D2C8), goat anti-IRSp53 (W-20), goat anti-Dia1 (C-20), and mouse anti-Arp3 ( 51 ) (Santa-Cruz); rabbit anti-Pak2 (2608) (Cell Signaling); mouse anti-Vav1 (39-2050) (Invitrogen); mouse anti-Lamp2 (human lysosome-associated membrane protein 2) (H4B4) (Santa Cruz Biotechnologies); anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase (HRP) (Dako); and anti-β-tubulin antibody coupled to HRP (ab21058) (Abcam).

Techniques: Transfection, Expressing, Western Blot, Flow Cytometry, Staining, Confocal Microscopy, SDS Page

Journal: Journal of Virology

Article Title: Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

doi: 10.1128/JVI.00469-15

Figure Lengend Snippet: Effect of Gag on Rac1 and RhoA activation and on F-actin content in T cells. Jurkat T cells were microporated with a control plasmid or pGag (HIV-1 Gag expression) or p8.2 (expression of Gag, Gag-Pol, and viral accessory proteins), together with the pCMV-GFP plasmid, which was used to normalize the microporation efficiency. (A and B) Effect of Gag on Rac1 and RhoA activation. Data shown are representative of data from three independent experiments. Rac1 (A) and RhoA (B) activation was measured by G-LISA. Data shown indicate the fold increases of GTPase activation regarding activation in Jurkat T cells expressing the control plasmid. * and ** indicate P values of <0.05 and 0.01, respectively. (C to E) Effect of Gag on F-actin content in Jurkat T cells as determined by flow cytometry. F-actin expression was measured by flow cytometry analysis in cells simultaneously fixed and permeabilized and then stained with phalloidin-Alexa 546. (C) Median percentages of cells expressing F-actin and standard deviations, which were calculated for Gag- or p8.2-microporated T cells compared to the GFP expression levels in control cells (microporated with pCMV-GFP), from seven independent experiments. (D) Data from a representative flow cytometry experiment. The continuous line represents F-actin fluorescence in T cells microporated with pGag or p8.2, and the discontinuous line is the control (T cells microporated with pCMV-GFP). * and ** indicate P values of <0.05 and 0.01, respectively. (E) Transfection efficiency was monitored by the percentage of GFP expression in each experiment. Values are averages of data from seven independent experiments. (F and G) Effect of Gag on F-actin content in Jurkat T cells, as determined by immunofluorescence imaging. Cells were transfected with pCMV-LacZ (control), pGag (encoding HIV-1 Gag), or p8.2 (encoding Gag, Gag-Pol, and accessory proteins). At 24 h posttransfection, Jurkat T cells were harvested, fixed, permeabilized, and stained with anti-MAp17 antibodies and phalloidin-Alexa 546. The left panels show images from a representative experiment. (G)The phalloidin intensity in Jurkat T cells under each condition was measured by image analysis (ImageJ) (n = 20 cells). The histogram shows the results obtained. * and ** indicate P values of <0.05 and 0.01, respectively.

Article Snippet: Immunoblottings were performed by using the following antibodies: anti-MAp17 and anti-CAp24 (NIH AIDS Reagent Program); HIV-1 p17 (VU47) rabbit and HIV-1 p24Gag monoclonal (24-4) mouse antisera, mouse anti-RhoA (ARH03), mouse anti-Rac1 (ARC03), and mouse anti-Cdc42 (ACD03) antisera (Cytoskeleton); rabbit anti-Wave2 (D2C8), goat anti-IRSp53 (W-20), goat anti-Dia1 (C-20), and mouse anti-Arp3 ( 51 ) (Santa-Cruz); rabbit anti-Pak2 (2608) (Cell Signaling); mouse anti-Vav1 (39-2050) (Invitrogen); mouse anti-Lamp2 (human lysosome-associated membrane protein 2) (H4B4) (Santa Cruz Biotechnologies); anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase (HRP) (Dako); and anti-β-tubulin antibody coupled to HRP (ab21058) (Abcam).

Techniques: Activation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Staining, Fluorescence, Transfection, Immunofluorescence, Imaging

Journal: Journal of Virology

Article Title: Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

doi: 10.1128/JVI.00469-15

Figure Lengend Snippet: Effect of Rho GTPase depletion on VLP production, Gag intracellular localization, and Gag membrane attachment in T cells. (A to E) Effect of Rac1, RhoA, and Cdc42 depletion on VLP production. Jurkat T cells were transfected with p8.2 (expression of Gag, Gag-Pol, and accessory viral proteins) and with the siRNA control or siRNA against Rac1, RhoA, or Cdc42. (A) Immunoblot analysis for detection of the HIV-1 proteins pr55Gag and CAp24 in cell lysates and in VLPs. Tubulin was used as a loading control. (B) Extracellular virus production measured by quantification of immunoblot images, i.e., the ratio between extracellular CAp24 and intracellular Pr55 Gag + CAp24. Bars show mean values and standard deviations resulting from three independent experiments. The statistical significances of differences were calculated by an unpaired t test. **, P value of <0.01; *, P value of <0.05. (C) Cell viability measured by flow cytometry analysis. (D) Quantification of Rho GTPase depletion after siRNA treatment. (E) Percent transfection measured by flow cytometry analysis. Bars show mean values and standard deviations resulting from three independent experiments. (F to J) Effect of Rac1, RhoA, and Cdc42 depletion on Gag intracellular localization. Jurkat T cells were transfected with p8.2 and with the siRNA control (F), siRNA against Rac1 (G), siRNA against RhoA (H), or siRNA against Cdc42 (I). Cells were fixed at 48 h posttransfection, permeabilized, stained for HIV-1 Gag, and analyzed by confocal microscopy. (J) Percentage of cells with each phenotype, calculated for 50 cells. Bars show mean values and standard deviations resulting from three independent experiments. (K and L) Effect of Rac1 or RhoA depletion on Gag cell membrane attachment. Jurkat T cells were microporated with p8.2 and with the siRNA control or siRNA against Rac1 or against RhoA. Cells were then lysed, and the PNS was used for membrane flotation assays. Intracellular proteins of each gradient fraction were loaded onto an SDS-PAGE gel. (K) The viral Gag and Lamp2 proteins were then revealed by immunoblotting, as indicated. (L) Rac1 and RhoA depletion by siRNA knockdown, in the PNS, shown by anti-Rac1 and anti-RhoA immunoblots, respectively.

Article Snippet: Immunoblottings were performed by using the following antibodies: anti-MAp17 and anti-CAp24 (NIH AIDS Reagent Program); HIV-1 p17 (VU47) rabbit and HIV-1 p24Gag monoclonal (24-4) mouse antisera, mouse anti-RhoA (ARH03), mouse anti-Rac1 (ARC03), and mouse anti-Cdc42 (ACD03) antisera (Cytoskeleton); rabbit anti-Wave2 (D2C8), goat anti-IRSp53 (W-20), goat anti-Dia1 (C-20), and mouse anti-Arp3 ( 51 ) (Santa-Cruz); rabbit anti-Pak2 (2608) (Cell Signaling); mouse anti-Vav1 (39-2050) (Invitrogen); mouse anti-Lamp2 (human lysosome-associated membrane protein 2) (H4B4) (Santa Cruz Biotechnologies); anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase (HRP) (Dako); and anti-β-tubulin antibody coupled to HRP (ab21058) (Abcam).

Techniques: Transfection, Expressing, Western Blot, Flow Cytometry, Staining, Confocal Microscopy, SDS Page