Sc 56971

Following transfection, cells were harvested daily at 1 to 7 dpi in RIPA buffer. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher) according to the manufacturer’s instructions. Proteins were separated on 4 to 12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes by either wet transfer (20% methanol [MeOH]) or Trans-Blot SD semidry transfer (mixed molecular weight). Membranes were probed with antibodies to viral proteins IE1 (Merck Millipore; MAB8131), IE2 (Merck Millipore; MAB8131), pp52 (Santa Cruz Biotechnology; sc-56971), and pp28 (Santa Cruz Biotechnology; sc-69749) and with antibodies to host proteins COPA (Santa Cruz Biotechnology; sc-398099), COPB2 (Novus Biologicals; NBP1-88651), ERC1 (Novus Biologicals; NBP1-88177), RAB4B (Santa Cruz Biotechnology; sc-26565), and actin (Abcam; ab8226). Secondary antibodies conjugated to IR800 dye (Li-Cor) were used, and blots were imaged by Li-Cor Odyssey infrared fluorescence. Quantification was done with Li-Cor Image Studio Lite software. Relative protein levels were calculated by first normalizing to the loading control (actin) and then by determining the ratio between the sample and its corresponding negative control.

Following transfection, cells were harvested at 0 to 7 DPI in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher) following the manufacturer’s protocol. The proteins were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes by wet transfer (20% methanol). The membranes were blocked with 5% milk in Tris-buffered saline (TBS) and probed with antibodies to HCMV IE1 and IE2 (MAB-8131; Merck Millipore; diluted 1/5,000), pp52 (sc-56971; Santa Cruz Biotechnology; 1/1,000), pp28 (sc-69749; Santa Cruz Biotechnology; 1/1,000), ASNS (catalog no. 14681-1-AP; Proteintech; 1/1,000), p70 S6 kinase (S6K) (Cell Signaling Technology; 2708; 1/1,000), phospho-Thr389 p70 S6K (catalog no. 9234; Cell Signaling Technology; 1/500), and β-actin (ab8227; Abcam; 1/2,500). Secondary antibodies conjugated to horseradish peroxidase (HRP) (Thermo Fisher) or IR800 and IR680 dye (Li-Cor) were used, and blots were imaged by Li-Cor Odyssey Fc imaging system. Quantification was performed with Li-Cor Image Studio Lite software.