Las 500 chemiluminescence imager

Total protein lysate was prepared in radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM sodium chloride, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris [pH 8.0]) supplemented with protease inhibitors and phenylmethylsulfonyl fluoride (PMSF). Protein concentration was quantified using a Bradford assay (Bio-Rad, Hercules, CA). Protein lysates were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes. ORF46 was detected using anti-ORF46 mouse sera described above, ORF59 was detected using affinity-purified chicken anti-peptide antibodies against MHV68 ORF59 (Gallus Immunotech, Fergus, Ontario, Canada) (65 (link)). Mouse sera harvested from MHV68-infected mice 28 dpi was used to detect lytic antigen. ORF65 antibody is a kind gift from Ren Sun (66 (link)). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody is from Sigma-Aldrich (St. Louis, MO). Detection was performed with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma), anti-rabbit IgG (Sigma) or anti-chicken IgY (Gallus) with an enhanced chemiluminescence reagent (ECL; Thermo Scientific). Data were collected with the LAS 500 chemiluminescence imager (GE Healthcare).

Infected Puro^R CRISPR-3T3 cells as described above were collected in RIPA lysis buffer (150 mM sodium chloride, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris [pH 8.0]) supplemented with protease inhibitors and PMSF. Protein concentration of the total protein lysate was quantified using Bradford assay (Bio-rad, Hercules, CA) and 40 μg were separated using 4–15% gradient SDS-PAGE and transferred to polyvinylidene fluoride membranes, followed by incubation with antibody to Cas9 (7A9-3A3, Cell Signaling Technology), ORF73 (mLANA) [37 (link)], GAPDH (Sigma-Aldrich, St. Louis, MO). Detection was performed with HRP-conjugated anti-mouse or anti-rabbit IgG (Sigma) by enhanced chemiluminescence reagent (ECL, Thermo Scientific). Data was collected with LAS 500 Chemiluminescence Imager (GE Healthcare).