Gapdh and β actin antibody

Protein samples were extracted from whole heart homogenates (n = 6/group, chosen randomly) and ADSCs as previously described [21 (link)]. Primary antibody against tumour necrosis factor-α (TNF-α) (R&D, Minneapolis, MN, USA), Akt1/2/3 (Santa Cruze, Santa Cruz, CA, USA), phospho-Akt (Santa Cruze), ERK (Cell Signaling), phosphor-ERK (Cell Signaling), signal transducers and activators of transcription 3 (STAT3; Cell Signaling), phosphor- STAT3 (Cell Signaling), Bcl-2 (Santa Cruze), Bax (Santa Cruze) and horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) were used according to manufacturers’ instructions. GAPDH and β-actin antibody (Cell Signaling Technology) was used to evaluate the amount of protein loaded in each sample (detailed in the supplementary material).

One million RPMI1788, Ramos or Daudi cells were lysed in 2× laemmli sample buffer (Bio-Rad) with β-mercaptoethanol and boiled at 95°C for 10 min. After removal of the insoluble fraction by centrifugation at 10,000 ×g for 10 min, protein samples were separated by SDS gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were stained with cleaved PARP, PARP, cleaved caspase-3, PUMA, XIAP and p53 antibodies (Cell Signaling Technology, MA, USA) at a dilution of 1: 1,000 or GAPDH and β-actin antibody (Cell Signaling Technology) at a dilution of 1: 5,000 at 4°C overnight. After overnight incubation in 4°C, HRP-conjugated secondary antibody was added. After washing with Tris-buffered saline and Tween 20, the hybridized bands were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).