Horseradish peroxidase conjugated anti mouse or anti rabbit antibody

hMSCs were incubated with 100 μM sirtinol for 24 h and then we collected lysates from cells exposed to 1% O₂ for 6 h.
Cells were lysed in buffer containing 20 mM Tris HCl, 100 mM NaCl, 10 mM MgCl₂, 1% NP40, 10% glycerol, 0.1 M NaF, 100 μM sodium vanadate, and protease inhibitors mixture (Roche LTD, GE). Equal amounts of supernatant were separated by SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (WhatmanProtran, GE Healthcare) and membranes were blocked with blocking buffer (TBS-Tween buffer containing 5% milk). Subsequently, the membranes were incubated with primary antibodies at 4°C overnight. After three washes for 10′ with TTBS buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, and 0.5% Tween-20), the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (1 : 10.000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature and then washed for 30 min with TTBS buffer. The resulting immunoblots were detected using Amersham ECL Plus (GE Healthcare).

The Ishikawa cells were treated with MHY2256 (0.2, 1, and 5 μM) or salermide (50 μM). After 48 h, the treated cells were collected and washed with cold Dulbecco’s phosphate-buffered saline (DPBS). Later, the cells were suspended in a PRO-PREP^TM protein extract solution (iNtRON, Seongnam, Korea) for 15 min. The total protein isolate and the protein concentration were measure with a protein assay kit (Bio-Rad, Hercules, CA, USA). The same amount of protein was loaded on a 6–15% sodium dodecyl polyacrylamide (PAGE) gel. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and the membranes were incubated with a TNA (50 mM Tris-HCl, pH 8; 100 mM NaCl, and 0.4 M l-arginine) buffer containing 5% skim milk for blocking for up to 1 h. Then, the membranes were incubated with different kinds of primary antibodies at 4 °C overnight. The following day, after washing the membrane with a TNA buffer for 1 h, it was incubated with a secondary antibody, horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA), for 1 h at room temperature, and was then further washed for 1 h. Finally, the membranes were developed with an enhanced chemiluminescence (ECL)-plus kit (Amersham Biosciences, Amersham, UK).

For the Western blotting experiments, protein electrophoresis was performed as described previously [52 (link)]. Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in TBST, and incubated overnight at room temperature using antibodies against the following: IL-10 (1:1000; Abcam, Cambridge, MA, USA), α-SMA (1:10,000; Sigma); Col-1 (1:1000; Abcam); fibronectin (FN; 1:1000; BD Biosciences, Franklin Lakes, NJ, USA); cleaved Cas3 (1:1000; LS Bio, Seattle, WA, USA); PARP (1:1000; Cell Signalling, Danvers, MA, USA); Bcl2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); Bcl-xL (1:1000; Santa Cruz); DR5 (1:1000; KOMA Biotech, Seoul, Korea); AIF (1:1000; Cell signalling); BiP (1:1000; Cell Signalling); CHOP (1:1000; Cell Signalling); IRE1 (1:1000; Cell Signalling); PERK (1:1000; Cell Signalling); and eIF2α (1:1000; Cell Signalling). After washing, membranes were incubated for 1 h at room temperature with a horseradish peroxidase conjugated anti-mouse or anti-rabbit antibody (1:5000; Santa Cruz). Specific proteins were visualized by enhanced chemiluminescence (PERKinElmer, Waltham, MA, USA). GAPDH (1:5000; Santa Cruz) and β-actin (1:10,000; Santa Cruz) were used as a loading control and for normalizing immunoblots, respectively, which were analyzed using NIH Image J.