fluorescein isothiocyanate 143 fitc conjugated donkey igg  (Jackson Immuno)

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: (A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an anti-CD46 antibody, an anti-CD147 antibody. A mouse IgG isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.

Article Snippet: If required, cells were secondary labelled with a fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG antibody (F5897, Merck).

Techniques: Knock-Out, Infection, Bacteria, Derivative Assay, Expressing, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: (A) Representative immunofluorescence images showing localisation of CD9 constructs. WT and CD9 -/- cells and those transfected with the CD9:TurboID fusion plasmid were fixed and treated with anti-CD9 (602.29) antibodies. Localisation was visualised using a FITC-conjugated secondary antibody. (B) The cell adhesion phenotype of CD9 is rescued with expression of the CD9:TurboID fusion protein in CD9 -/- cells. Calcein AM labelled cells were allowed to adhere to fibronectin coated wells for 60 minutes or 24 hours. (C) Meningococcal and staphylococcal infection phenotypes are rescued with expression of CD9:TurboID fusion protein in CD9 -/- cells. Cells were infected with meningococci (MC58) or staphylococci (SH1000) for 60 minutes at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by cfu. n>3, mean + SEM, One-Way ANOVA.

Article Snippet: If required, cells were secondary labelled with a fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG antibody (F5897, Merck).

Techniques: Immunofluorescence, Construct, Transfection, Plasmid Preparation, Expressing, Infection, Bacteria