Pp2a activity kit

DMEM, RPMI, trypsin-EDTA 0.25% and penicillin-streptomycin (PS) was purchased from GE Healthcare Life Sciences Hyclone Laboratories (Pittsburg, PA, USA). Fetal bovine serum (FBS) was received from Gibco (Thermo Fisher Scientific Inc., Walthem, MA, USA). ApoScan^TM annexin V-FITC was obtained from BioBud (Cat. No.: LS-02-100, Seongnam, Gyeonggi-do, Korea). MTT cell viability assay kit EZ-CYTOX was acquired from Do-GenBio Co. Ltd. (Seoul, Korea). PP2A activity kit was purchased from Millipore Corporation (Billerica, MA, USA). Antibodies against AKT (9272), pAKT (Thr308) (13038), Caspase-3 (9662), BAX (2772), and PARP (9542), and Bcl-2 (3498) were provided from Cell Signaling Technology (Danvers, MA, USA). β-actin (sc-47778), cytochrome-c (sc-13156), ERK 1/2 (sc-514302), pERK (Thr202/Tyr204) (sc-136521), and HRP-conjugated anti-mouse and anti-rabbit antibodies were received from Santa Cruz Biotechnology (Dallas, TX, USA). SK1 (ab71700) and SK1 (ab264042) antibodies were acquired from Abcam (Cambridge, UK). Protein marker, protease, and phosphatase inhibitor cocktail were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ECL Solution for western blotting imaging was received from Millipore Corporation (Burlington, MA, USA).

HEK cells were purchased from ATCC. Antibodies against PR65a and PP2A catalytic subunit were from Santa Cruz Inc and Millipore Inc. Antibody against EF1A was from Cell signaling Inc. PP2A activity kit was purchased from Millipore Inc. QuickChange™ mutagenesis kit was from Stratagene.
Male 250–300 g Dahl salt-sensitive (S) or Dahl salt-resistant (R) rats were purchased from Harlan Sprague Dawley. All animal studies were done in accordance with University of Illinois at Chicago Institutional Animal Care and Use Committee (IACUC). Rats were maintained on a 8% NaCl diet ad lib.

We used a PP2A activity kit (Milipore, Billerica, MA) to determine cellular PP2A activity as described previously^{39 (link)}. In brief, the effects of CANA on affecting PP2A were tested in Huh7 and PP2Ac-immunoprecipitant protein complex harvested from Huh7 cells. After cells exposed to indicate treatments, cell lysates were prepared within a low-detergent lysis buffer, and a specific reaction buffer containing 750 mM phosphopeptide substrate was added. The whole reaction took place for 10 min at 30 °C. The amounts of free phosphate after CANA treatment were determined by the optical density revealed by malachite dye at 650 nm.