Antibody against beclin 1

MSCs were quickly harvested and homogenized on ice in modified RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (vol/vol) Triton X-100, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 1 mM EDTA, 50 mM N-ethylmaleimide, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF and 1 μg ml⁻¹ each of aprotinin, leupeptin and pepstatin. The cell extracts (~200 μg of total protein) were incubated with an antibody against Beclin-1 (2.5 μl, Cell Signaling Technology) at 4 °C overnight. Then, protein-G agarose beads (40 μl, Beyotime Biotechnology, Shanghai, China) were added, and the mixture was incubated at 4 °C for 3 h. The agarose beads were collected, washed, and resuspended in 60 μl of sample buffer containing 50 mM Tris-HCl, pH 7.6, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 10 mM DTT and 0.2% bromophenol blue. Afterwards, the samples were boiled for 5 min and analyzed via western blot.

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Hycolon (Logan, UT, USA). Opti-MEM medium was from Gibco BRL (Carlsbad, CA, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). Rap (10 nM), 3-MA (5 mM), iRTX (1 μM), capsaicin and compound C (10 μM) were obtained from Sigma-Aldrich (St. Louis, MO, USA). GFP-LC3-expressing plasmid (pEGFP-LC3) was from Cell Biolabs (San Diego, CA, USA). Annexin V-FITC Apoptosis Detection Kit was from Beyotime (Beijing, China). Caspase-Glo 3/7 kit was from Promega (Southampton, UK). Antibodies targeting Atg7 (1 : 1000), LAMP-1 (1 : 1000), β-actin (1 : 2000), OPN (1 : 1000), Atg7 siRNA (sc-41448) and the scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against LC3 (2 μg/ml) was from Novus Biologicals (Littleton, CO, USA). Antibody against beclin-1 (1 : 1000) was from Cell Signaling Technology (Beverly, MA, USA). Antibody targeting α-SMA (1 : 1000) was from Abcam (Burlingame, CA, USA). The enhanced chemiluminescence (ECL) substrate was purchased from Pierce (San Diego, CA, USA).