Fostriecin

Simvastatin, rifampicin, pravastatin sodium salt hydrate, phosphatase inhibitor cocktail 2, phosphatase inhibitor cocktail 3, ANTI-FLAG M2 Affinity Gel, ANTI-FLAG M2-Peroxidase (HRP) and monoclonal ANTI-FLAG M1 antibodies were purchased from Sigma-Aldrich (St. Louis, MO); restriction endonucleases and DNA-modifying enzymes from New England Biolabs (Beverly, MA); cOmplete Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland); okadaic acid from EMD Millipore (Billerica, MA); fostriecin, sanguinarine chloride and GSK 650394 from Tocris Bioscience (Bristol, UK); anti-PXR antibody from Perseus Proteomics Inc. (Tokyo, Japan); anti-SGK2 antibody and anti-PP2Cα (D18C10) XP antibody from Cell Signaling (Danvers, MA); anti-PP2Ac antibody from BD Biosciences (Franklin Lakes, NJ); anti-p-SGK antibody (Thr256), anti-β-actin antibody (C4), PP1α (C-19) antibody, anti-RXR (ΔN 197) antibody. goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, donkey anti-goat IgG-HRP, normal mouse IgG and tautomycin from Santa Cruz Biotechnology (Santa Cruz, CA).

A TnT-coupled rabbit reticulocyte lysate (RRL) (Promega) in vitro translation and capsid assembly system was used to express the WT or mutant HBc proteins, as described previously [29 (link),62 ]. In vitro translated proteins were incubated in the 1X NEB Buffer 3 containing the 1X EDTA-free Protease Inhibitor (cOmplete) and 1X RNasin Plus Ribonuclease Inhibitor (Promega, N2611) for 16 hrs at 37°C to allow HBc dephosphorylation by endogenous RRL phosphatases. Alkaline Phosphatase, Calf Intestinal (CIAP) (NEB, M0290S) (10U) was added to some reactions as a positive control for dephosphorylation. To prevent dephosphorylation, the 1X Phosphatase inhibitor cocktail 2&3 (Sigma, P5726&P0044), or specific inhibitors of cellular phosphatases, including okadaic acid (Sigma, O9381) (1 nM, 10 nM, 100 nM) and fostriecin (10 nM, 20 nM,40 nM, 80 nM) (Tocris, 1840) was added following translation and before the dephosphorylation reaction. HBc proteins were then resolved by NAGE, SDS-PAGE, or Phos-tag SDS-PAGE and detected by western blot analysis using the indicated HBc antibodies.