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.
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.
