Phospho thr 389 p70s6k

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.

Proteins were extracted from SKOV3 cells treated with rfhSP-D for 24 and 48 h (21 (link)) and separated on a 10% v/v SDS-PAGE. The separated proteins were then electrophoretically transferred onto a nitrocellulose membrane (Thermo Scientific) in Wet-Transfer Buffer. The membrane was incubated in TBS containing 5% w/v dried milk powder and 0.1% v/v Tween Tween-20, for 1 h at room temperature to block non-specific binding. The membrane was then incubated with primary anti-human SP-D polyclonal antibodies (raised in rabbit), caspases 3 and 9, and phospho (Thr389) p70S6K (Cell Signaling Technology) at 4°C overnight. The membranes were washed in TBS + 0.1% Tween-20 (3 times, 15 min each) before incubation with the secondary goat anti-rabbit IgG-HRP-conjugated antibody for 1 h at room temperature. Proteins were visualized as previously described (21 (link)).