Journal:

Article Title: Identification of a Cytoplasmic Targeting/Retention Signal in a Retroviral Gag Polyprotein

doi:

Figure Lengend Snippet: Determination of intracytoplasmic capsid formation and myristylation of mutant Gag polyproteins. (A) Western blot showing intracytoplasmic immature capsid assembly in mock-infected cells (lanes 1 and 2) and in SV-T2 cells expressing wild-type MoMuLV (lanes 3 and 4), MLV/D-CTRS (lanes 5 and 6), and MLV/C-CTRS (lanes 7 and 8). The cells were lysed in 1% Triton X-100-containing ICAP buffer and then fractionated into soluble (S; lanes 1, 3, 5, and 7) and pelletable (P; lanes 2, 4, 6, and 8) fractions by centrifugation. Gag polyproteins (Pr65gag) in each fraction were immunoprecipitated with rabbit anti-Gag antiserum and then visualized by Western blot analysis. Ig, heavy chains of rabbit immunoglobulin molecules. (B) The Gag polyprotein complexes in the pellet fraction of MLV/D-CTRS cells were purified through a continuous 30 to 50% (wt/wt) linear sucrose gradient. The fractions were collected from bottom to top, diluted with PBS, and ultracentrifuged to pellet high-molecular-weight particles. Particle-associated Gag polyproteins were detected by Western blot assay. The density of fraction 5 is 1.20 g/ml. (C) Myristylation of mutant Gag polyproteins was determined by labeling cells with [3H]leucine (lanes 1 to 4) or [3H]myristic acid (lanes 5 to 8). Radiolabeled Gag molecules of wild-type MoMuLV (lanes 2 and 6), MLV/D-CTRS (lanes 3 and 7), and MLV/C-CTRS (lanes 4 and 8), as well as mock-infected cells (lanes 1 and 5), were immunoprecipitated with rabbit anti-Gag antibodies.

Article Snippet: To establish cell lines containing integrated wild-type or mutant proviral DNA, semiconfluent monolayers of murine BALB/c 3T3 fibroblast SV-T2 cells (ATCC CCL-163.1) that are negative for MuLV were transfected with viral DNAs linearized with Fsp I as described previously ( 33 ).

Techniques: Mutagenesis, Western Blot, Infection, Expressing, Centrifugation, Immunoprecipitation, Purification, Molecular Weight, Labeling

Journal: PLOS Biology

Article Title: A flexible loop in the paxillin LIM3 domain mediates its direct binding to integrin β subunits

doi: 10.1371/journal.pbio.3002757

Figure Lengend Snippet: ( A ) 293T cells were transiently cotransfected with a CEACAM3-ITGB3 (ITGB3) fusion construct together with GFP or the indicated GFP-labelled proteins. Cells were seeded on poly-L-lysine and incubated for 1 h with Pacific Blue–labelled Neisseria gonorrhoeae (Ngo; blue) to cluster ITGB3 (stained red with α-CEACAM antibody). Recruitment of GFP-fused proteins to the clustered integrin β3 tail is indicated by white arrowheads. Bars represent 2 μm. ( B ) Stable GFP-paxillin expressing Flp-In 3T3 cells were transiently transfected with RFP-tagged paxillin LIM1-4 or RFP-tagged paxillin LD1-5 domains. Displacement of GFP-paxillin by RFP-LIM1-4, but not RFP-LD1-5, is visible in boxed and enlarged areas. ( C ) GFP-Paxillin localization at FAs in presence of LIM1-4 or LD1-5 domains as in (B) was evaluated by measuring the GFP fluorescence intensity. Shown are mean values of GFP-paxillin intensity from 3 independent experiments. Total number of analysed FAs is given in brackets under each sample. Error bars represent 5 and 95 percentiles. Significance was calculated using one-way ANOVA, followed by Bonferroni multiple comparison test (*** p < 0.0001, ns = not significant). The data underlying this panel can be found in . ( D ) GFP-FAK expressing MEFs were transiently transfected with RFP-paxillin full-length or RFP-LIM1-4. Displacement of GFP-FAK by RFP-LIM1-4, but not RFP-Paxillin, is visible in boxed and enlarged areas. ( E ) Calculation of GFP-FAK intensity at FAs was performed as in (C) n = 360 FAs per sample from 3 independent experiments. Significance was calculated using one-way ANOVA, followed by Bonferroni multiple comparison test (*** p < 0.0001). The data underlying this panel can be found in . ( F ) In vitro pulldown using recombinant Strep-tag integrin β1 or β3 cytoplasmic tails and recombinant talin1 F3 domain, full-length kindlin2 or paxillin LIM2/3 domain fused to His-SUMO, or His-SUMO only as negative control.

Article Snippet: Flp-In 3T3 murine fibroblasts (Thermo Fisher Scientific) were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and 1% nonessential amino acids.

Techniques: Construct, Incubation, Staining, Expressing, Transfection, Fluorescence, Comparison, In Vitro, Recombinant, Strep-tag, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Oligomerization, F-actin Interaction, and Membrane Association of the Ubiquitous Mammalian Coronin 3 Are Mediated by Its Carboxyl Terminus

doi: 10.1074/jbc.m205136200

Figure Lengend Snippet: FIG. 3. Localization of Hcoronin 3 in different cell lines. A–C, primary human skin fibroblasts; D–F, Swiss 3T3 fibroblasts serum starved overnight and left untreated (D) or incubated with 100 nM PMA (15 min, E) or 10 g/ml insulin (30 min, F). Arrowhead, accumulation of coronin 3 at lamellipodia. Cells were fixed in methanol and incubated with mAb K6-444 specific for coronin 3, followed by fluorescein isothio- cyanate-conjugated goat anti-mouse IgG antibody. G–I, nonconfluent Swiss 3T3 cells left untreated (G) or incubated with 10 M cytochalasin D (CytD, 30 min, H and I). Cells were fixed in paraformaldehyde and stained for coronin 3 (G and H) and F-actin (TRITC-phalloidin, I). Arrowheads, localization of coronin 3 (H) and actin accumulation (I). J and K, Swiss 3T3 cells transfected with the plasmids indicated and paraformaldehyde-fixed 16 h post-transfection. Only cells expressing the Myc-tagged GTPases as identified by staining with rabbit anti-Myc antibody followed by Alexa 568-coupled goat anti-rabbit IgG are shown. Bars, 50 m.

Article Snippet: Swiss 3T3 murine fibroblasts were obtained from the DSMZ (Braunschweig, Germany) and cultured under standard conditions in Dulbecco’s modified Eagle’s medium with 1 g/liter glucose, 10% fetal calf serum, penicillin/streptomycin, and L-glutamine.

Techniques: Incubation, Staining, Transfection, Expressing