Protease inhibitor cocktail

HF cells were plated in 60-mm dishes, allowed to grow to confluence, and incubated for 3 days prior to serum starvation for 24 h. Cells were infected with HCMV AD 169 at an MOI of 1. A confluent cell population and an asynchronous cell population were utilized as negative and positive controls, respectively, for Grasp phosphorylation. For the asynchronous cell population, cells were plated at a subconfluent density and, 24 h later, were serum starved for 24 h. Medium containing serum was then added, and cells were returned to the incubator for 22 h prior to collection. Cells were lysed in HEPES lysis buffer containing 0.5% NP-40 on ice, and the lysate was incubated with a protease inhibitor cocktail (Research Products International, Prospect, IL) and/or a phosphatase inhibitor cocktail (PIC; RPI, Prospect, IL). Cell lysates were incubated with calf intestinal phosphatase (New England Biolabs, Ipswich, MA) at a concentration of 10,000 U/µg lysate in NEB buffer 3 at 37°C for 3 h to remove phosphates from protein. SDS loading buffer (5×) was added to the lysate prior to boiling and loading on gels. Equivalent protein loading was quantified using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA).

Protein was isolated from parasites by resuspending harvested parasites in RIPA lysis buffer supplemented with a protease inhibitor cocktail (Research Products International Corp P506001). Samples were then sonicated using a QSonica Q800R3 at 50% amplitude for 2 min. Insoluble material was pelleted and removed. Protein concentrations of lysates were determined using a BCA protein assay kit (Thermo Fisher Scientific 23227), and 50 ug of protein was used for Western blotting. Protein samples were separated by SDS-PAGE in 4%–15% Bis-Tris gels with MOPS buffer and transferred to nitrocellulose membrane. Membranes were blocked in 5% non-fat milk and incubated in primary and secondary antibodies diluted in 5% non-fat milk. The following antibodies were used: anti-myc-HRP (Santa Cruz sc-40) diluted 1:100, anti-HA (Roche 27573500) diluted 1:2,000, anti-rat-HRP (GE NA935) diluted 1:2,000, anti-p30 (SAG1) (Invitrogen MA183499) diluted 1:2,000, and anti-mouse-HRP (GE NA931) diluted 1:2,000. Pierce Enhanced Chemiluminescence (ECL) detection reagent (Thermo Fisher Scientific 32109) and a BioRadV3 Chemidoc Imager were used to visualize blots.

For mass spectrometry assays, cells were hormone deprived for 8 d and treated for 1 h with 100 μM biotin +/− 1 nM E2. For immunoblot assays, cells were hormone deprived for 4 d and treated with 100 μM biotin +/− 1 nM E2 × 24 h. Cells were rinsed with PBS and lysed with base lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 0.5% Triton X-100, 5 mM β-glycerophosphate, 2 mM NaF, 2 mM molybdate) with 1:500 Protease Inhibitor Cocktail (Research Products International, Mt Prospect, IL, USA). Lysates were centrifuged at 17,000× g for 10 min at 4 °C. Protein concentrations were determined by BCA assay (Pierce), and concentrations were equalized across samples. Strep-Tactin Sepharose 50% suspension beads (IBA Lifesciences, Goettingen Germany) were used for biotinylated protein pulldown. Lysate was incubated with beads for 3 h at 4 °C. Beads were washed and protein was eluted in 100 μL 2% SDS, 50 mM Tris pH 8.0, and 5 mM biotin at 85 °C for 15 min. Eluted protein was analyzed by LC-MS/MS (described below) or immunoblot.