Journal: Cell Communication and Signaling : CCS

Article Title: Zika virus: mapping and reprogramming the entry

doi: 10.1186/s12964-019-0349-z

Figure Lengend Snippet: Subcellular localization of ZIKV in Rab35-depleted cells upon NH 4 Cl treatment. ( a ) Confocal analysis of ZIKV localization 1 h p.i. in NH 4 Cl-treated Vero cells. ZIKV envelope protein is visualized in green, actin cytoskeleton stained in red to show the cell boundaries and nuclei are shown in blue. Scale bar = 10 μm. ( b ) Western blot analysis of the efficiency of siRNA-dependent Rab35 silencing (Rab35 expression in Vero cells compared to GAPDH expression in these cells). M – BlueStar prestained protein marker; Ø – normal non-transfected Vero cells. ( c ) Graph representing the percent of ZIKV particles present inside cells related to the total number of ZIKV particles, assessed from confocal images of siRNA-transfected and all control Vero cells infected with ZIKV H/PF/2013 in the presence of 50 mM NH 4 Cl. v + NH 4 Cl - ZIKV-infected, NH 4 Cl-treated, non-transfected cells; v + sh + NH 4 Cl - ZIKV-infected, NH 4 Cl-treated, sham transfected cells; v + sc + NH 4 Cl - ZIKV-infected, NH 4 Cl-treated, scrambled siRNA transfected cells; v + si + NH 4 Cl - ZIKV-infected, NH 4 Cl-treated, Rab35-specific siRNA transfected cells; vØ - ZIKV-infected, NH 4 Cl-untreated, non-transfected cells; mock - mock-infected, NH 4 Cl-untreated, non-transfected cells. The data is presented as the mean ± SD. To determine the significance of differences between compared groups, single-factor analysis of variance (ANOVA) was applied. P values < 0.05 were considered significant. One asterisk (*) identifies adjusted P values between 0.01 and 0.05, two asterisks (**) identify adjusted P values between 0.01 and 0.001, three asterisks (***) identify adjusted P values between 0.001 and 0.0001

Article Snippet: To visualize host cell proteins, slides were incubated with primary antibodies against clathrin, EEA1, Rab7, LAMP1, Rab11, Rab27 and Rab35 [goat anti-clathrin HC (RRID:AB_2083170), rabbit anti-EEA1 (RRID:AB_2277714) and rabbit anti-Rab7 (RRID:AB_2175483) polyclonal antibodies from Santa Cruz Biotechnology, Poland, rabbit anti-Rab11A (RRID:AB_2173458) polyclonal antibody from Proteintech, UK, rabbit anti-Rab27A monoclonal antibodies from Cell Signaling Technology, Poland, rabbit anti-Rab35 polyclonal antibody from Novus Biologicals, Poland, rabbit anti-LAMP1 (RRID:AB_2134611) polyclonal antibody from Thermofisher Scientific, Poland] diluted 1:100 in 3% BSA in PBS, together with anti-ZIKV antibodies.

Techniques: Staining, Western Blot, Expressing, Marker, Transfection, Control, Infection

Journal: Diabetes

Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

doi: 10.2337/db15-1563

Figure Lengend Snippet: Hsp20 promotes exosome biogenesis via interacting with Tsg101. A: Diagram of exosome biogenesis/release pathway. B: Overexpression of Hsp20 in the heart increased the expression levels of major mediators involved in exosome generation (n = 3). *P < 0.05 vs. WT. α-Actin was used as a loading control. C: Cardiomyocytes isolated from adult WT or TG mouse hearts were successfully cultured. D: The exosome concentration was increased in the culture supernatants of TG cardiomyocytes (n = 3 wells). *P < 0.05 vs. WT. Similar results were observed from three different hearts. E: A scheme for protein-protein interaction analysis. F: Recombinant protein Tsg101 coated on a plate dose-dependently captured the Hsp20 protein, whereas Rab11a/b, Rab35, and control protein BSA coated did not effectively arrest the Hsp20 protein. G and H: Coimmunoprecipitation of heart homogenates with Hsp20 antibody or Tsg101 antibody showed that Hsp20 directly interacted with Tsg101. Ab, antibody; CM, cardiomyocyte; IP, immunoprecipitation; OD, optical density.

Article Snippet: The source of antibodies and dilutions used were as follows: rabbit anti-CD63 (sc-15363), rabbit anti-CD81 (sc-9158), and rabbit anti-Tsg101 (Santa Cruz Biotechnology); mouse anti–p-Akt (Ser473), mouse anti-Akt, rabbit anti-survivin, anti-Rab11a, anti-Rab11b, and anti-Rab35 antibodies (Cell Signaling); and rabbit anti-Hsp22, anti-Hsp27, anti-Hsp60, anti-Hsp70, anti-Hsp90, and anti–αB-crystallin (Affinity BioReagents).

Techniques: Over Expression, Expressing, Control, Isolation, Cell Culture, Concentration Assay, Recombinant, Immunoprecipitation

Journal: Nature Communications

Article Title: Negative regulation of EGFR signalling by the human folliculin tumour suppressor protein

doi: 10.1038/ncomms15866

Figure Lengend Snippet: ( a ) U2OS cells were transiently transfected with empty vector, FLCN WT, Rab7A WT, Rab7A Q67L, Rab7A T22N or combinations, as indicated. Co-purified complexes were detected by immunoblotting with antibodies recognizing FLCN or Rab7A, as indicated. ( b ) 293T cells were transiently transfected with an empty vector, FLAG-FLCN WT, FLAG FLCN S62/73A, FLAG FLCN S62/73E, HA-Rab7A WT or combinations, as indicated. Co-purified complexes were detected by immunoblotting with antibodies recognizing FLCN or HA (Rab7A), as indicated. ( c ) U2OS cells were transfected with empty vector, or FLCN WT and HA-GFP-Rab7A. Co-localization was identified by confocal microscopy (yellow, indicated by arrows in the insert corresponding to the region outlined by the white box). Scale bars, 5 μm (upper panel) and 10 μm (lower panel). ( d ) HA-Rab7A and FLAG-tagged FLCN WT proteins were purified from transfected 293T cells with anti-HA (Rab7A) or anti-FLAG antibodies. The amount of inorganic phosphate released due to GTPase activity was measured by GTPase colorimetric assay kit. GTPase activity was quantified in four independent experiments, and data are represented as mean±s.e.m. Significance (*) was conferred at P <0.05, ANOVA and Tukey’s Multiple Comparison post-tests. ( e ) Same as in d , except FLCN WT and FLCN K508R (untagged) were immunoprecipitated with an anti-FLCN antibody from the lysates of transfected 293T cells. The data are represented as mean±s.e.m. and were collected in two independent experiments. * indicates statistical significance, P <0.05, ANOVA and Tukey’s Multiple Comparison post-tests. ( f ) GST-FLCN WT or GST-FLCN C9 mutant proteins were incubated with HA-Rab7A, purified from transfected 293T cells. The GAP activity was measured as in d , e . Data are represented as mean±s.e.m., n =5. * indicates statistical significance, Two-tailed paired t -test, P =0.0037. ( g ) U2OS cells were transiently transfected with an empty vector, FLCN WT, Rab7A WT, Rab7B or combinations, as indicated. Co-purified complexes were identified by immunoprecipitation followed by immunoblot, as indicated. ( h ) Same as in g , except U2OS cells were transiently transfected with Rab7B, Rab35 or Rab8A, as indicated. ( i ) Same as in g , except U2OS cells were transiently transfected with Rab9A.

Article Snippet: Rab7B-Myc-DDK (RC202283) and Rab35-Myc-DDK (RC201932) in pCMV6-Entry were purchased from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Purification, Western Blot, Confocal Microscopy, Activity Assay, Colorimetric Assay, Immunoprecipitation, Mutagenesis, Incubation, Two Tailed Test