Chemiscope 3400

Western blot analysis was performed according to the procedures outlined by Hu
et al. and Larson-Casey et al.^{2 (link),21 (link)} Briefly, after
electrophoresis, the proteins were transferred to polyvinylidene difluoride
membranes (Millipore, Bedford, MA, USA). The membranes were incubated with
primary Ab at 4℃ for 10 h and then with the secondary Ab for 2 h at room
temperature. The primary Abs [occludin, claduin-1, zonula occludens-1 (ZO-1),
light chain 3-I (LC3-I), LC3-II, Parkin, (PTEN-induced putative kinase) Pink1,
VADC, β-actin] were purchased from Santa Cruz Technology Inc. (Santa Cruz, CA,
USA). The secondary Ab was HRP-conjugated anti-rabbit Ab (Cell Signaling
Technology, Danvers, MA, USA). Western blot was detected with an enhanced
chemiluminescence detection kit (Amersham, Arlington Heights, IL, USA),
photographed by a ChemiScope 3400 (Clinx Science Instruments, Shanghai, China)
and analyzed using Quantity One software. β-Actin and VDAC were used as internal
controls, and exhibited no differences bwteen the groups. The relative
abundances of intestinal target proteins and mt target proteins were expressed
as target protein/β-actin, target protein/VDAC protein ratio, respectively. The
protein expression of all samples was expressed as fold changes, calculated
relative to the control group.

HEK293 cells were co-transfected with plasmids encoding the full-length CD146, netrin-1 or the truncation mutants using Fugene HD (Roche). 48 h post transfection, the cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 0.1% SDS, 0.5% deoxycholate, 0.1% NP-40, 1 mM PMSF, protease inhibitor cocktails). Then the cell lysates were incubated with the control mIgG, anti-CD146 mAb AA1 or anti-netrin-1 mAb for 4 h at 4 °C, and immunoprecipitation was carried out using protein G sepharose beads (Santa Cruz). The cell lysates and precipitates were analyzed by western blotting using chemiluminescence imaging system (ChemiScope 3400; Clinx, China).

Total protein extracts were mixed with sample buffer (125 mM Tris-HCl, pH 6.8, 2% SDS w/v, 20% glycerol v/v, 20 µg/µL bromophenol blue, and 5% β-mercaptoethanol), boiled for 10 minutes, separated by electrophoresis on a 10% SDS-PAGE gel, and blotted onto a BioTrace™ nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA). The membranes were blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20% and 5% nonfat dry milk at room temperature for 1 hour and incubated with antibodies to PARP-1 (46D11; rabbit mAb #9532; Cell Signaling Technology, Danvers, MA, USA and mouse β-actin (A5316; Sigma-Aldrich Co., St. Louis, MO, USA). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody in blocking solution at 4°C. The immune complex was visualized using Pierce™ ECL Western Blotting Substrate, enhanced chemiluminescent luminol substrate (#32106; Thermo Fisher Scientific) according to the manufacturer’s instructions. The chemiluminescent signal was captured by a ChemiScope 34|00 (Clinx; Science Instruments Co., Ltd., Shanghai, China). Reactive bands were quantified by densitometry using ImageJ software.