Complete protease inhibitor tablet

For cytoplasmic and nuclear fractionation, AML12 cells were grown to 100% confluency, washed twice with ice-cold PBS and incubated with buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, pH 7.9, PMSF 50 µg ml⁻¹, Na-Orthovanadate 1 mM, DTT 1 mM, NP-40 (0.6%), HALT-Phosphatase inhibitor (Thermoscientific) 1:1000, complete Protease-inhibitor tablet (Thermoscientific)) on ice for 15 min. Cells were collected and centrifuged for 5 min at 14,000 × g. Supernatant (cytoplasmic fraction) was collected and saved. The pellet was processed further for nuclear extraction by washing with buffer A without NP-40, followed by resuspension in buffer C (10 mM HEPES, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.9, PMSF 50 µg ml⁻¹, Na-Orthovanadate 1 mM, DTT 1 mM, NP-40 (0.6%), HALT-Phosphatase inhibitor (Thermoscientific) 1:1000, complete Protease-inhibitor tablet (Thermoscientific)) and incubation on ice for 15 min, vortexing every minute. Fraction was centrifuged at 14,000 × g for 8 min and supernatant (nuclear fraction) collected.

The Akt1/2 knockout HCT116 colon cancer cell line was given as a gift from Dr Bert Vogelstein (Johns Hopkins University; Ericson et al., 2010 (link)). Cells were cultured in McCoy’s 5A (Gibco) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco) at 37°C and 5% CO₂. When the cell confluence reached around 70% in six-well plates, the medium was changed with McCoy’s 5A (Gibco) having 5% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco), and the cells were transfected with 1.5 μg of pcDNA3.1-Flag-HA-Akt plasmid or 1.5 μg (3.0 μg for Y18A mutant) of pcDNA3.1-Myr-HA-Akt plasmid complexed with 3 μL Lipofectamine 3000 (Invitrogen) and 3 μL P3000 reagent (Invitrogen) in Opti-MEM medium (Gibco) at 37°C and 5% CO₂. Seventy-two hours after transfection, the cells were washed with cold PBS and lysed by adding 150 μL RIPA buffer (Cell Signaling Technology) containing ×1 complete protease inhibitor tablet (Thermo Fisher Scientific) and ×1 PhosStop tablet (Roche), and 1 mM PMSF for 10 min at 4°C. Thirty μg of total protein (BCA assay) was loaded on an SDS-PAGE gel. Membrane transfer and western blotting were carried out as described above with 1:1000 dilution for primary antibodies: anti-Akt, anti-pT308 Akt, anti-Foxo1, anti-Foxo3a, and anti-pT24 Foxo1/pT32 Foxo3a, anti-GAPDH (Cell Signaling Technology).

HBV Cp was expressed in E. coli BL21(DE3) cells (T7 Expression strain, New England Biolabs), expressed from a pET28b plasmid. Induction with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at an optical density (OD₆₀₀) of ~0.6 was followed by growth for 20 h at 21 °C. Cells were lysed using a Soniprep 150 and clarified by spinning at 11,000 × g for 1 h. NCPs were then pelleted by centrifugation at 120,000 × g for 14 h. Pellets were resuspended in 20 mM HEPES (pH 7.5), 250 mM NaCl, 5 mM dithiothreitol (DTT) and applied to a prepacked Captocore 700 column (GE Life Sciences). Fractions containing NCPs were pooled and precipitated with 40% (w/v) ammonium sulfate. NCPs were dissociated into Cp dimers by dialysis into 1.5 M guanidinium chloride (GuHCl) as previously described^{12 (link),38 (link)}. All steps after sonication were performed in the presence of complete protease inhibitor tablets (1 tablet per 500 mL buffer, Thermofisher Scientific). Cp dimer concentration was determined by the absorbance at 280 nm (ε₂₈₀ of Cp₂ = 55,920 L mol⁻¹ cm⁻¹). Cp fractions with an A_260/280 ratio of ≤0.65 were used in assembly assays.