Sc 13560

Cells on coverslips were stained for MFF and cytochrome c(CYTO c). After mitochondrial staining, the cells were incubated with primary antibodies against MFF (ab81127, Abcam) or cytochrome c (sc-13560, Santa Cruz) and then incubated with rhodamine- or FITC-conjugated secondary antibodies (Invitrogen). The coverslips were counterstained with 46-diamidino-2-phenyl indole and imaged under a confocal microscope TCS SP5 (Lecia, Solms, Germany).

Protein extracts from the heart and liver tissue were obtained by homogenization in lysis buffer (100 mg/mL) as mentioned previously [1 (link), 26 (link)]. The protein concentrations were determined by Lowry protein assay and the samples were electrophoresed in a 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were then transferred to PVDF membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked using 5% non-fat milk for 1 h. Monoclonal primary antibodies were diluted 1:1000 in antibody binding buffer (TBS) and used for hybridization overnight (4 °C) with the following antibodies ANP, phosphor-Akt, β-Actin, Bax, BNP, Cytochrome C, phosphor-GATA4, PGC1α (sc-20158, sc-7985, sc-47778, sc-526, sc-18818, sc-13560, sc-32823-R, sc-13067, Santa Cruz); phosphor-AMPK, Cle-Caspase-3, phosphor-FOXO3a, Sirt1 (#2535, #9664, #9466, #9475s, Cell Signaling, The Netherlands). Following hybridization with appropriate secondary antibodies the membranes were washed in for 10 min thrice. The blots were detected in chemiluminescent detection using ECL with Fujifilm LAS-3000 (GE Healthcare Life Sciences).