Western Blot Analysis
The protocol of Western blot analysis was described previously. In
brief, AMDC seeded into 6-well plates at a density of 1 ×
105 cells/well and treated by TC with varying
concentrations described above (0, 6, 12 μg/mL). After 48 h incubation,
total proteins in cells were extracted using radioimmunoprecipitation
assay (RIPA buffer high) lysis buffer (60 mM Tris-HCl, pH 6.8, 5%
glycerol, 2% SDS) (Solarbio, R0010, Beijing, China), PMSF 100 Mm
(Solarbio, P0100, Beijing, China), Aprotinin from bovine lung (Solarbio,
9087-70-1, Beijing, China), Protein Phosphatase Inhibitor
(All-in-one,100x) (Solarbio, P1260, Beijing, China) with the proportion
of 100:1:1:1 on ice and quantified using BCA protein concentration
determination kit (Beyotime, P0012, Beijing, China) according to the
manufacturer’s instruction. Subsequently, proteins were in
polyacrylamide gels, transferred onto polyvinylidene difluoride (PVDF)
membranes (Millipore, NY, USA), and incubated with primary antibodies at
4 °C overnight. After washing with Tris Buffered Saline Tween (TBST;
Solarbio, T1086, Beijing, China) for 3 times, the PVDF membranes were
then incubated with horseradish peroxidase-conjugated secondary
antibodies at room temperature for 1 h. The GAPDH antibody (1:10000;
Proteintech, Cat No.:10494-1-AP, Wuhan, China) signal was used as a
loading control. Finally, the bound antibodies were detected with
Immobilon Western Chemiluminescent HRP Substrate (Millipore, NY, USA).
Images were obtained with Bio-Rad ChemiDocTM XRS system (Bio-Rad,
Shanghai, China). The intensity of the target proteins blots was
quantified by Image J software version 1.8.0.