Journal: bioRxiv

Article Title: Identification of A Disintegrin and Metalloproteinase 9 domain (ADAM9) required in the early stages of encephalomyocarditis virus infection

doi: 10.1101/491068

Figure Lengend Snippet: WT and ADAM9 KO HeLa cells were transduced with retroviral vectors with wild-type (WT) murine ADAM9 (mADAM9), catalytically inactive mutant ADAM9 (E>A), cytoplasmic tail deleted (ΔCT) ADAM9 constructs, or GFP control vectors. Transduced cells were selected and cloned by limiting dilution, and expression of mADAM9 was confirmed by western blot analysis. (A) WT, KO and rescue cell lysates were prepared in RIPA buffer and run on 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 3% BSA and incubated with ADAM9 antibodies. Upper panel, rabbit anti-human ADAM9 (Cell signaling #2099) that detects an epitope in the intracellular domain of human ADAM9 and cross-reacts with mouse ADAM9. Middle panel, goat anti-mouse ADAM9 (R&D systems AF949) that detects the extracellular domain of murine ADAM9, but not human ADAM9. Lower panel, anti-Actin (Santa Cruz sc-1616) which detects both human and murine ϟ-Actin. Bands were visualized using HRP and ECL reagent. Upper panel: WT but not KO cells expressed human ADAM9. Middle panel: Rescue but not KO cells expressed murine ADAM9. Lower panel: Actin loading control. (B) WT clones, ADAM9 KO clones and rescued ADAM9-expressing clones were infected with EMCV or CVB3 at varying MOI and incubated at 37 °C for 24 h. Viability of EMCV-infected and CVB3-infected clones was measured by CellGlo ATP luminescence. (C) EMCV replication was quantified in infected culture supernatants by plaque assay using BHK-21 cells. Neither the functional sequence of the ADAM9 metalloproteinase domain nor the cytoplasmic tail are required for EMCV infection. ***, P <0.0001, KO vs. WT; KO vs. rescue.

Article Snippet: 3% BSA blocking buffer), anti-goat IgG HRP (1:2000 dilution, R&D Systems HAF109), and anti-rabbit IgG HRP (1:5000 dilution Vector Labs PI1000).

Techniques: Transduction, Mutagenesis, Construct, Clone Assay, Expressing, Western Blot, SDS Page, Incubation, Infection, Plaque Assay, Functional Assay, Sequencing