Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: Analysis of vimentin GlcNDAz crosslinks. ( A ) 293T cells transfected with vimentin-myc-6xHis were treated with DMSO vehicle or GlcNDAz for 24 hr, treated with vehicle, 100 mM 5SGlcNAc, or 50 mM Thiamet-G for an additional 24 hr, and then UV-crosslinked. Lysates were analyzed by fluorescent IB (V9 antibody). Nucleoporin-62 is a positive control for GlcNDAz crosslinking. GAPDH is a loading control. ( B ) 293T cells transfected with vimentin-myc-6xHis were treated with DMSO vehicle or GlcNDAz (48 hr) and UV light (or not) as indicated. Lysates were analyzed by IB (D21H3 antibody). Vimentin-myc-6xHis and endogenous vimentin crosslink similarly. ( C ) 293T cells were transfected with vimentin-myc-6xHis for 48 hr, treated with DMSO vehicle or GlcNDAz as in , and UV-crosslinked. Crosslinked and uncrosslinked vimentin populations were purified from lysates by tandem anti-myc IP and nickel affinity chromatography. Crosslinks were visualized by colloidal Coomassie stain (left) and IB (D21H3 antibody) (right).

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Transfection, Positive Control, Control, Purification, Affinity Chromatography, Staining

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: ( D ) Peptide coverage map from GlcNDAz-crosslinked complexes of vimentin purified in (C; see ). Found peptides are highlighted in yellow. ( E ) Top protein IDs from vimentin crosslinks. Full proteomics results are available for download as a Scaffold (.sf3) file on the eLife web site . Free software to view Scaffold files is available at http://www.proteomesoftware.com/products/scaffold/download/ . ( F ) 293T cells were GlcNDAz-crosslinked as in (A; see ) and subjected to differential extraction as in Figure 1D (left). Then, the 8M urea fractions from DMSO or GlcNDAz samples were divided, half of each sample was exchanged into non-denaturing IP buffer, and samples were analyzed by vimentin IB (V9 antibody) (right). ( G ) HeLa cells were treated with DMSO vehicle or 1% IDPN for 1 hr, fixed, stained for vimentin (D21H3 antibody), and analyzed by confocal microscopy. ( H ) 293T cells were treated with DMSO (vehicle) or GlcNDAz for 48 hr and UV light as indicated. Lysates were normalized for protein amount and analyzed by fluorescent IB (V9 antibody). n = 4.

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Purification, Software, Extraction, Staining, Confocal Microscopy

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: Successful CRISPR/Cas9-mediated deletion of vimentin was verified by actin-normalized qPCR ( A ) and IB (D21H3 antibody) ( B ). mRNA from WT and vimentin knockout mouse embryonic fibroblasts (MEFs) serve as positive and negative controls for the qPCR assay in ( A ). Single vimentin −/− clones (HeLa, sgRNA 1 clone 4; 293T, sgRNA 3 clone 4) were used to construct vimentin-reconstituted cell lines. ( C ) Parental (i.e., un-manipulated) HeLa cells (control) and vimentin −/− HeLa cells reconstituted with either empty vector or WT vimentin-mEmerald were lysed and analyzed by fluorescent anti-vimentin IB (V9 antibody) (left). Quantification of the fluorescence intensities (right) demonstrates that vimentin-mEmerald expression level is indistinguishable from that of endogenous vimentin. n = 2. n.s., not significant, Student’s t-test. ( D ) Vimentin −/− HeLa cells reconstituted with empty vector (control) or WT or mutant vimentin-mEmerald constructs were sorted by flow cytometry to obtain the top one-third of mEmerald-expressing cells and then used in all subsequent experiments. ( E ) Parental HeLa cells were transfected with empty vector or WT vimentin-mEmerald for 24 hr, fixed, permeabilized, and imaged for total vimentin (D21H3 antibody immunofluorescence) and vimentin-mEmerald (mEmerald tag fluorescence).

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: CRISPR, Knock-Out, Clone Assay, Construct, Control, Plasmid Preparation, Fluorescence, Expressing, Mutagenesis, Flow Cytometry, Transfection, Immunofluorescence

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: 293T cells were transfected with vimentin-myc-6xHis constructs as indicated and treated with either vehicle (DMSO) or 12 µM MG132 for 13 hr. Lysates were analyzed by fluorescent IB (V9 antibody for vimentin).

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Transfection, Construct

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet:

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Stable Transfection, Expressing, Recombinant, Plasmid Preparation, Sequencing, Functional Assay, Virus, Cloning

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were infected with C. trachomatis L2 for 24 hrs and then fixed and stained with antibodies to GBF1 (red) and BIG1 (green). Bacteria and host DNA were detected using DAPI (blue). The cis and trans polarity of the Golgi was maintained in C. trachomatis L2-infected cells. N, host nucleus. *, inclusion. Scale bar = 5 µm. (B) HeLa cells were transfected with Arf1-GFP for 18 hrs, infected with C. trachomatis L2 for 24 hrs in the absence or presence of 10 µM BFA, and then fixed and stained with antibodies to GBF1 (red). Enlargements of boxed regions are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. Arf1-GFP localized to the region between two closely apposed inclusions (white arrow) and to a thin rim around the inclusion (red arrow) whereas GBF1 was excluded from these regions. *, inclusion. Scale bar = 5 µm. (C) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (D) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then labeled with BODIPY FL-Ceramide to visualize SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (E) HeLa cells were infected with C. trachomatis for 24 hrs, treated with 10 µM BFA or GCA during the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (F) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then analyzed for progeny formation as described in . Values (mean ± standard error) are shown as percentage of control siRNA-treated samples. No significant decrease in progeny formation was observed. IFU, inclusion forming units.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Infection, Staining, Transfection, Western Blot, Labeling

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were treated with the indicated siRNA for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to 14-3-3β (green) to identify the inclusion membrane and GBF1 (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to breaks in the inclusion membrane where the bacteria are released into cytoplasm in GBF1-depleted cells. Inclusions formed in BIG1 and/or BIG2 depleted cells remain intact. (B) HeLa cells were depleted of GBF1 for 3 days, infected with C. trachomatis L2 for 24 hrs, then fixed and stained with antibodies to MOMP (green) to identify bacteria and vimentin (red). Bacteria and host DNA were detected using DAPI (blue). The exposure time for each filter set for all images was identical. White arrows point to the region on the inclusion that is devoid of vimentin staining and where bacteria are released into the cytoplasm. N, host nucleus; *, inclusion. MOMP, C. trachomatis major outer membrane protein. Scale bar = 5 µm.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Infection, Staining

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells transfected with CERT-GFP for 18 hrs were left uninfected or infected with C. trachomatis L2 for 24 hrs. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm. (B) HeLa cells transfected with CERT-GFP were infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Bacteria and host DNA were detected using DAPI (blue). Enlargements (inset) of boxed regions are shown to the right. *, inclusions. Scale bar = 5 µm. (C–E) HeLa cells were transfected with CERT-GFP and HcRedVAP-A for 18 hrs and infected with C. trachomatis L2 for (C) 2, (D) 8, or (E) 24 hrs. (C) Cells were stained with DAPI to visualize the nascent inclusions (red arrows). (D and E) Enlargements (inset) of boxed regions are shown to the right. At 8 and 24 hpi, CERT-GFP and HcRedVAP-A colocalize on the inclusion membrane and exhibit a patchy distribution. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). N, host nucleus; *, inclusions. Scale bar = 5 µm, except with insets from panels B, C, and D where scale bar = 2.5 µm.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Transfection, Infection, Staining

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: HeLa cells were infected with C. trachomatis L2, treated with the indicated concentration of HPA-12 at 1–24 hpi, and then (A) fixed and stained with antibodies to MOMP (red) and with DAPI (blue) to visualize bacteria or (B) analyzed for progeny formation. Values (mean ± standard error) are shown as percentage of DMSO treated samples. Data are representative of 3 independent experiments. ***p<0.001 compared to DMSO treated cells (ANOVA). (C) HeLa cells were transfected with CERT-GFP and HA-CKIγ2 for 18 hrs, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to HA (red). The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Ectopic expression of HA-CKIγ2 decreased inclusion size but did not affect CERT-GFP recruitment to the inclusion membrane. Scale bar = 5 µm. (D) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (E) HeLa cells were depleted of CERT for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. CERT depletion reduced SM accumulation in the Golgi. Scale bar = 5 µm, (F) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to MOMP (green) to identify the inclusion. Bacteria and host DNA were detected using DAPI (blue). The exposure time for all images was identical. CERT depletion reduced inclusion size. Red arrows point to inclusions. Scale bar = 5 µm. (G) HeLa cells were depleted of CERT for 3 days, infected with C. trachomatis L2 and analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. CERT depletion significantly reduced inclusion size and progeny formation. Data are representative of 2 independent experiments. ***p<0.001 for CERT siRNA-treated cells compared to control siRNA-treated cells (ANOVA). N, host nucleus. IFU, inclusion forming units. Scale bar = 5 µm.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Infection, Concentration Assay, Staining, Transfection, Expressing, Western Blot, Labeling

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were transfected with CERT-GFP, CERT (D324A)-GFP, or CERT (G67E)-GFP, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Mutation of the PI4P binding (G67E) or VAP-A binding (D324A) domains did not affect CERT-GFP recruitment to the inclusion. Scale bar = 5 µm. (B) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 50 µM Exo1 (Arf1 inhibitor) for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT-GFP localization to the inclusion was unaffected by Arf1 inhibition. Scale bar = 5 µm. (C) HeLa cells expressing CERT-GFP were infected with C. trachomatis L2, treated with 5 µM HPA-12 for 1–24 hpi, and then fixed and stained with antibodies to MOMP (red) to identify bacteria or to p230 (red) to identify the trans -Golgi. The exposure time for each filter set of all images was identical. Inhibition of CERT transfer and/or ceramide binding activity by HPA-12 treatment resulted in loss of CERT accumulation on the inclusion membrane. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). Scale bar = 5 µm. N, host nucleus; *, inclusion.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Transfection, Infection, Staining, Mutagenesis, Binding Assay, Expressing, Inhibition, Activity Assay

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells were infected with C. trachomatis L2 for 24 hrs, treated with 5 µM HPA-12 for the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM accumulation by the inclusion. As a control for decreased SM acquisition by the inclusion, cells were also treated with 10 µM BFA or 25 µg/ml D609. The exposure time of all images was identical. Dashed red lines demarcate inclusions. The residual fluorescence likely represents Golgi staining. Scale bar = 5 µm. (B) Quantitation of SM acquisition following treatment with HPA-12, D609, or BFA. Values (mean ± standard error) are shown as percentage of mean fluorescence intensities relative to DMSO-treated samples. ***p<0.001 (ANOVA). HPA-12, D609, and BFA-treated cells displayed a significant decrease in fluorescence intensity of the inclusion and its contents compared to DMSO-treated samples.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Infection, Labeling, Fluorescence, Staining, Quantitation Assay

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) HeLa cells co-transfected for 18 hrs with CERT-GFP and C-terminally 3xFLAG-tagged SMS1/SMS2 (upper 2 rows) or SMS1-V5 and C-terminally 3xFLAG-tagged SMS2 (bottom row) were infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with antibodies to FLAG (red) and/or to V5 (green). Single channel images of uninfected cells are shown to the right. Enlargements of the boxed regions (inset) in infected samples are shown to the right of infected set. The exposure time for each filter set of all images was identical. Images shown are maximum intensity projections of confocal z-stacks (0.4-µm slices). CERT and SMS1 localization at the inclusion are distinct while CERT and SMS2 partially overlap. SMS2 localization partially overlaps with SMS1 around the inclusion, however SMS2 also localizes to the inclusion. At longer exposure times, SMS2 plasma membrane localization is evident. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (B) HeLa cells transfected with SMS1-V5 (green) or SMS2-V5 (green) infected with C. trachomatis serovar D for 24 hrs and then fixed and stained with antibodies to IncA (red) to identify the inclusion membrane. Enlargements of the boxed regions (inset) are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. SMS2 but not SMS1 partially overlaps with IncA on the inclusion. Scale bar = 5 µm, except the insets where scale bar = 2.5 µm. (C) HeLa cells transfected for 18 hrs with CERT-GFP, SMS1-V5, or SMS2-V5 were infected with C. trachomatis L2, treated with 10 µM BFA or Nocodazole for 1–24 hpi, and then fixed and stained with antibodies to V5 (green) and to p230 (red) to identify the trans -Golgi. BFA and Nocodazole disrupted SMS1 localization around the inclusion but had no effect on SMS2 or CERT localization at the inclusion. Images represent a single z slice from confocal images. Scale bar = 5 µm. N, host nucleus; *, inclusion.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Transfection, Infection, Staining

Journal: PLoS Pathogens

Article Title: Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

doi: 10.1371/journal.ppat.1002198

Figure Lengend Snippet: (A) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (B) HeLa cells were depleted of SMS1 or SMS2 for 3 days and then labeled with BODIPY FL-Ceramide to analyze SM accumulation in the Golgi. The exposure time of all images was identical. SMS1 but not SMS2 depletion reduced BODIPY FL lipid accumulation in the Golgi. Scale bar = 5 µm. (C and D) HeLa cells were depleted of SMS1 or SMS2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then fixed and stained with an antibody to MOMP (red). Bacteria and host DNA were detected using DAPI (blue). (D) SMS1 and SMS2-depleted cells were analyzed for inclusion size and progeny formation. Values (mean ± standard error) are shown as percentage of control siRNA samples. SMS1 and SMS2 depletion reduced inclusion size and production of infectious progeny. Data are representative of 2 independent experiments. ***p<0.001, all samples compared to control siRNA treatment (ANOVA). N, host nucleus; red arrows point to inclusions. IFU, inclusion forming units. Scale bar = 5 µm.

Article Snippet: Antibodies were obtained from the following sources: mouse anti- Chlamydia FITC conjugate (Meridian Diagnostics), goat anti- C. trachomatis MOMP (Cortex Biochem), mouse anti-GAPDH (Chemicon), mouse anti-GBF1 (BD Transduction Laboratories), rabbit anti-BIG1 (Santa Cruz), rabbit anti-BIG2 (Bethyl Laboratories, Inc.), chicken anti-CERT (Sigma), rabbit anti-14-3-3β (Santa Cruz), mouse anti-vimentin (Sigma), mouse anti-FLAG (Sigma), rabbit anti-V5 (Sigma), mouse anti-HA (Covance), rabbit anti-p58 (Sigma), mouse anti-ceramide IgM (Clone15B4), rabbit anti-calnexin (Cell Signaling), rabbit anti-goat IgG horseradish peroxidase (HRP) (Calbiochem), goat anti-rabbit IgG HRP (Amersham Biosciences), goat anti-mouse HRP (Amersham Biosciences), donkey anti-goat Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), chicken anti-mouse 594 (Molecular Probes), goat anti-mouse IgG1 Alexa 594 (Molecular Probes), donkey anti-goat Alexa 488 (Molecular Probes), Texas red-conjugated donkey anti-chicken IgY (Jackson Laboratories), and donkey anti-rabbit Alexa 488 (Molecular Probes).

Techniques: Western Blot, Labeling, Infection, Staining

Journal: Rheumatology (Oxford, England)

Article Title: A potential role for chlamydial infection in rheumatoid arthritis development

doi: 10.1093/rheumatology/kead682

Figure Lengend Snippet: Prevalence of anti-MOMP IgG antibodies in serum of RA-FDR defined by their preclinical status

Article Snippet: To explore the robustness of our primary exposure, we performed a nested case–control study in a subset of RA-FDRs for a secondary exposure of interest, the serology against C. trachomatis major outer membrane protein (MOMP) IgG (ELISA, Vircell, Granada, Spain, cat. no. G/M1017).

Techniques: