integrin a5  (Santa Cruz Biotechnology)

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

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: Molecular modeling of extracellular ISG15-α5β1 integrin interaction . A) Crystal structure of α5β1 integrin bound with fibronectin (PDB ID: 7NWL). B) RGD motif binding site at the integrin α5β1 integrin headpiece. RGD residues of fibronectin are shown as licorice, while β-propeller of α5 and βI domain of β1 subunits are shown as cartoon representation. C) Crystal structure of ISG15 (PDB ID: 3RT3). D) Docked orientation of ISG15 with integrin α5β1. E) Residues of ISG15 binding with the RGD motif binding site of α5β1 integrin are shown as orange-colored sticks. F) Superposition of fibronectin RGD residues with the residues of ISG15. Arg99 of ISG15 is positioned similar to the arginine (RGD) of fibronectin and Glu80 occupies the aspartic acid (RGD) binding site. G) Integrin α5β1’s activation-specific interactions with ISG15. Please make (F) and (G) clearer.

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: Binding Assay, Activation Assay

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: Schematic model showing antiviral pro-ISGylation activity of extracellular ISG15 via a type-I interferon (IFN) independent mechanism. Virus infection will trigger intracellular buildup of cellular unconjugated ISG15 protein, which will be utilized for ISGylation. A portion of unconjugated ISG15 will be released from infected cells. Extracellular ISG15 will interact with the cell surface integrin molecules of uninfected bystander cells (paracrine action) to trigger FAK-dependent intracellular ISG15 induction and subsequent ISGylation via an IFN independent mechanism. Since ISGylation possesses antiviral activity, it will confer an antiviral state in the uninfected bystander cells to ensure reduced viral infectivity during subsequent infection of these cells. Apart from paracrine action, extracellular ISG15 can also act on infected cells (autocrine action) for optimal antiviral response.

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: Activity Assay, Virus, Infection