Western blot analysis was performed on whole cell extracts (30 µg lysate) of NIH/3T3 (Lane 1), RAW 264.7 (Lane 2), 3T3-L1 (Lane 3), A549 (Lane 4) and MCF7 (Lane 5). The blot was probed with Anti-ISG15 (Mouse) Recombinant Rabbit Monoclonal Antibody (Product # 703132, 1:5000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution). A ~17 kDa band corresponding to free form of ISG15 was observed in only the mouse cell lines tested.
For immunofluorescence analysis, RAW 264.7 cells were fixed and permeabilized for detection of endogenous ISG15 (Mouse) using Anti-ISG15 (Mouse) Recombinant Rabbit Monoclonal Antibody (Product # 703132, 1:100) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Product # R415, 1:300) Panel a-d) shows representative un-treated cells that were stained for detection and localization of ISG15 (Mouse) protein (green) with less signal compared to panel e-h) clearly demonstrating enhanced cytoplasmic localisation of ISG15 (Mouse) in RAW 264.7 cells treated with IFN alpha (500u/mL, 24h). The images were captured at 60X magnification.
Supplier Page from Thermo Fisher Scientific for ISG15 Antibody