Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: ( A ) Schematic representation of Crk proteins under investigation. ( B ) WB of HeLa cell extracts using anti-CrkI/II MoAb and anti-CrkL Ab and the Odyssey imaging system to show expression levels of the corresponding proteins, or ( C ) to show decreased Crk expression in infected cells pretreated with siRNA. As a loading control the blots were probed with anti-actin monoclonal Ab (MoAb) or anti-tubulin Ab (upper bands). ( D ) Confocal fluorescence images of HeLa cells pretreated with siRNA against CrkI/II and CrkL or control siRNA and infected with preactivated EPEC for 2 h at an MOI of 3. Actin was stained with TRITC-phalloidin (red), while bacteria were stained with DAPI (blue). Arrows point at pedestals and bacteria. The scale bar represents 20 μm. ( E ) Quantitation of the number of pedestals on infected HeLa cells pretreated using siRNA with two oligonucleotides against CrkI/II and CrkL (black bar) compared to control oligonucleotide treated cells (white bar). Quantitation was done by counting the number of pedestals on 100 cells. Data in the graph show mean ± standard deviation (SD) for three independent experiments. The difference between groups was statistically significant based on Students t -test analysis; **, p<0.01.

Article Snippet: The following commercial Abs were used: anti-Flag M2 mouse MoAb (Sigma), anti-GFP polyclonal and anti-GST monoclonal Abs (BD Biosciences and B14 MoAb from Santa Cruz Biotechnologies); anti-Myc tag 4A6 mouse MoAb (Merck); anti-CrkI/II mouse MoAb clone 22 (BD Biosciences Pharmingen); anti-CrkII rabbit polyclonal Ab (H53, Santa Cruz Biotechnology); and anti-CrkL rabbit polyclonal Ab (Ab C-20, Santa Cruz Biotechnology).

Techniques: Imaging, Expressing, Infection, Fluorescence, Staining, Quantitation Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: ( A ) WB with anti-CrkL Ab in WT and CrkI/II-deficient MEFs to show lower CrkL levels in cells treated using siRNA with an oligonucleotide against CrkL than in cells treated with a control oligonucleotide (lower bands). The levels of injected Tir effector were assessed by WB with anti-Tir MoAb. As a loading control, blots were probed with anti-actin MoAb. ( B ) Immunofluorescence images of WT and CrkI/II-deficient MEFs in which CrkL expression was inhibited by siRNA; cells were infected with preactivated EPEC for 3 h at an MOI of 3. Actin was stained red with TRITC-phalloidin, while EPEC was stained blue using anti-LPS Ab followed by Alexa 405-conjugated goat anti-mouse secondary Ab. Confocal images were merged using Leica software. Insets are 4× digital zoom images. ( C ) Quantitation of the number of pedestals and the ratio of pedestals to bacteria on WT and CrkI/II-deficient cells treated using siRNA with a scrambled control (Ctr.) oligonucleotide (white and dark-grey bars, respectively) or with an oligonucleotide to reduce CrkL expression (grey and black bars, respectively). The number of pedestals was quantified based on counts with 100 cells. The ratio of pedestals to bacteria was quantified by counting the number of adhered bacteria with or without pedestals on 50 cells; the resulting ratio was expressed as a percentage. The graph shows mean ± standard deviation (SD) for three independent experiments. The indicated groups differed significantly based on Student's t -test. *, p<0.05; **, p<0.01.

Article Snippet: The following commercial Abs were used: anti-Flag M2 mouse MoAb (Sigma), anti-GFP polyclonal and anti-GST monoclonal Abs (BD Biosciences and B14 MoAb from Santa Cruz Biotechnologies); anti-Myc tag 4A6 mouse MoAb (Merck); anti-CrkI/II mouse MoAb clone 22 (BD Biosciences Pharmingen); anti-CrkII rabbit polyclonal Ab (H53, Santa Cruz Biotechnology); and anti-CrkL rabbit polyclonal Ab (Ab C-20, Santa Cruz Biotechnology).

Techniques: Injection, Immunofluorescence, Expressing, Infection, Staining, Software, Quantitation Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: ( A ) WB with anti-CrkII Ab in WT and CrkL-deficient MEFs to show lower CrkII levels in cells treated using siRNA with an oligonucleotide against CrkI/II than in cells treated with control (Ctr.) oligonucleotide. The blot was probed with anti-CrkL Ab as a control for cell genotype, and with anti-actin MoAb as a loading control. The levels of Tir effector are shown. ( B ) Fluorescence images of WT and CrkL-deficient MEFs in which CrkI/II expression was inhibited by siRNA; cells were infected with preactivated EPEC at an MOI of 3. Actin was stained red with TRITC-phalloidin, while EPEC was stained blue using DAPI. Images were merged using Leica software. Insets are 4× digital zoom images. ( C ) Quantitation of the number of pedestals on WT and CrkL-deficient cells treated for siRNA using a scrambled control (Ctr.) oligonucleotide (white bar) or an oligonucleotide to reduce CrkI/II expression (black bar). Quantitation was done by counting the number of pedestals on 100 cells. The graph shows mean ± standard deviation (SD) for three independent experiments. The indicated groups differed significantly based on Student's t -test. **, p<0.01.

Article Snippet: The following commercial Abs were used: anti-Flag M2 mouse MoAb (Sigma), anti-GFP polyclonal and anti-GST monoclonal Abs (BD Biosciences and B14 MoAb from Santa Cruz Biotechnologies); anti-Myc tag 4A6 mouse MoAb (Merck); anti-CrkI/II mouse MoAb clone 22 (BD Biosciences Pharmingen); anti-CrkII rabbit polyclonal Ab (H53, Santa Cruz Biotechnology); and anti-CrkL rabbit polyclonal Ab (Ab C-20, Santa Cruz Biotechnology).

Techniques: Fluorescence, Expressing, Infection, Staining, Software, Quantitation Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: HeLa cells were serum-starved for 16 h prior to infection with preactivated EPEC for 1, 2 or 3 h at an MOI of 45. The basal level of phosphorylation of Crk proteins was visualized in uninfected cells (- EPEC). ( A ) CrkII or IgG isotype control immunoprecipitates were probed by WB with a phosphospecific Ab against phospho-Tyr221 in CrkII (phospho-CrkII) to show induction of phosphorylation. The blot was also probed with anti-CrkI/II MoAb to show total levels of CrkII. The ratio of phospho-CrkII to non-phosphorylated CrkII in a representative experiment is shown. ( B ) Statistical analysis of the ratio of phospho-CrkII to CrkII signals with respect to the basal level in uninfected cells using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. ***, p<0.001. ( C ) WBs with a phosphospecific Ab against phospho-Tyr207 in CrkL to show the induction of phosphorylation, and with anti-CrkL Ab to show total levels of CrkL. The ratio of the phospho-CrkL to CrkL in a representative experiment is shown. ( D ) Statistical analysis of the ratio of phospho-CrkL to CrkL levels with respect to the basal level at 0 h using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. *, p<0.05. ( E ) Immunofluorescence images of HeLa cells that were starved for 16 h prior to infection with preactivated EPEC for 3 h at an MOI of 15. Immunofluorescence staining was done using phosphospecific Abs against Tyr221 in CrkII (phospho-CrkII) or Tyr207 in CrkL (phospho-CrkL) followed by Alexa 488-conjugated goat anti-rabbit secondary Ab (green). Actin was stained red using TRITC-phalloidin; bacteria were stained blue using DAPI. Pictures were taken on a confocal microscope and images from one section are shown, together with 4× digital zoom images (insets). Images were merged using Leica software.

Article Snippet: The following commercial Abs were used: anti-Flag M2 mouse MoAb (Sigma), anti-GFP polyclonal and anti-GST monoclonal Abs (BD Biosciences and B14 MoAb from Santa Cruz Biotechnologies); anti-Myc tag 4A6 mouse MoAb (Merck); anti-CrkI/II mouse MoAb clone 22 (BD Biosciences Pharmingen); anti-CrkII rabbit polyclonal Ab (H53, Santa Cruz Biotechnology); and anti-CrkL rabbit polyclonal Ab (Ab C-20, Santa Cruz Biotechnology).

Techniques: Infection, Immunofluorescence, Staining, Microscopy, Software