Calcineurin

The protein samples were extracted from the LV of rats with the procedures as previously described [25 (link)]. The protein concentration was determined using the BCA method as recommended by the manufacturer. After boiled for 5 min., the protein samples were fractionated by SDS-PAGE (10–15% polyacrylamide gels) and transferred to PVDF membrane (Millipore, Bedford, MA, USA). The samples were blocked with milk powder for 1 hr at room temperature and then incubated with primary antibodies K_V1.4, K_V1.5, K_V2.1, K_V4.2, K_V4.3 (Alomone Labs, Jerusalem, Israel) as well as calcineurin (Santa Cruz Biotechnology Inc., Santa Cruz, CA USA) and NFATc3 (Santa Cruz Biotechnology Inc.) at 4°C overnight. After washing, the membranes were incubated with a secondary antibody for 1 hr at room temperature. Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area × OD) for each group and normalizing to GAPDH as an internal control.

Betulinic acid was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), and penicillin-streptomycin F-actin, Alexa Four 488 phalloidin, dichlorofluorescin diacetate (DCFH-DA), DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Doxorubicin, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), primary antibodies for p38, ERK, JNK, p-ERK1/2, p-p38, NfκB p65, Bcl-2, Bax, β-actin (sc-47778), β-MHC, MLC-2v, p-GATA-4, GATA-4, Calcineurin, HDAC, LaminB, and HRP conjugated secondary antibodies raised against mouse, rabbit and goat were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NFAT-c3, Caspase-3, Caspase-9, Bax, and α-tubulin were purchased from Cell signaling technology (Danvers, MA, USA). ANP and BNP were purchased from ABCam (Danvers, MA, USA).