Journal: bioRxiv
Article Title: Extracellular ISG15 triggers ISGylation via a type-I interferon independent mechanism to regulate host response during virus infection
doi: 10.1101/2024.07.05.602290
Figure Lengend Snippet: A) Cell lysate from A549 cells incubated with purified recombinant ISG15 protein (rISG15, 5µg/mL) or vehicle for 4h at 4°C were immune-precipitated (IP) with ISG15 antibody and subsequently immuno-blotted (IB) with α5 integrin antibody. WCL were also blotted with α5 integrin and actin antibodies. B) Biotinylated purified α5β1 integrin protein (5.7 mM) or biotinylatedvehicle (control was incubated with rISG15 protein (0.1 µM) for 16h at 4°C. Following incubation, biotinylated molecules were precipitated with avidin-agarose beads and the avidin-agarose bound complex was immune-blotted (IB) with ISG15 antibody. C) WCL from A549 cells incubated with either rISG15 (1µg/mL) or vehicle for 15 minutes (15m) or 30m were subjected to immunoblotting with phospho-FAK antibody that detects Tyr925 phosphorylation of activated FAK. WCL was also immunoblotted with FAK and actin antibodies. D) A549 cells were treated with rISG15 (1µg/mL) in the presence of either vehicle control or FAK inhibitor (FAKi, 20 µM). WCL collected from these cells were subjected to immunoblotting with ISG15 antibody to detect unconjugated monomeric form of ISG15. E) A549 cells were treated with rISG15 (1µg/mL) in the presence of either vehicle control or FAK inhibitor (FAKi, 20 µM). WCL collected from these cells were subjected to immunoblotting with ISG15 antibody to detect ISGylated proteins. F) Wild type (WT) and α5 integrin KO (ITGA5 KO) HAP1 cells were treated with rISG15 (5ug/mL). WCL collected from these cells were subjected to immunoblotting with ISG15 antibody. G) A549 cells were treated with rISG15 (2µg/mL) in the presence of either control peptide or RGD peptide (200 µM). Cells were also treated with rISG15 in the absence of any peptide (untreated cells). WCL collected from these cells were subjected to immunoblotting with ISG15 antibody to detect ISGylated proteins. H) A549 cells were treated with rISG15 (2µg/mL) in the presence of either control peptide or RGD peptide (200 µM). Cells were also treated with rISG15 in the absence of any peptide (untreated cells). WCL collected from these cells were subjected to immunoblotting with ISG15 antibody to detect unconjugated monomeric form of ISG15.
Article Snippet: Membranes were blocked in 5% milk with PBS-T for 5 hours at room temperature and blotted with primary antibodies against ISG15 generated as described previously , Rhodopsin C9 (Invitrogen, catalog no. MA1-722), S1 (Sinobiological), RFP (ThermoFisher Scientific, catalog no. MA5-15257), pFAK(Y397) (Cell signaling, catalog no. 8556P), FAK (Cell signaling catalog no. 13009S), a5 integrin (Proteintech Catalog no.10569-1-AP), and actin (Bethyl Laboratories, Inc., catalog no. A300-491A) overnight at 4°C.
Techniques: Incubation, Purification, Recombinant, Control, Avidin-Biotin Assay, Western Blot, Phospho-proteomics
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