After thorough washing with buffer A, the membrane was probed for caspase-9.
Primary antibody was diluted in blocking buffer and incubated with the membrane.
Each primary antibody (anti-PDGFR-α, anti-PDGFR-β and anti-phosphorylated PDGFR-β, Cell Signaling Technology, Inc. Germany) was diluted in blocking buffer and incubated with the membrane overnight at 4°C.
After 3 times washes with TBST buffer, the membrane was incubated with anti-mouse-HRP and goat anti-rabbit-HRP for 30 minuates, respectively.
After washing with stripping buffer, the membrane was reprobed with GAPDH (1 : 5000, Kangchen, China), using ultra enhanced chemiluminescence western blotting detection reagents.
After washing with buffer, the membranes were blotted with corresponding secondary antibodies conjugated with horseradish peroxidase.
After several washes with washing buffer, the membrane was transferred to DAB assay reagents (Catalog No. P0203, Beyotime, China) for chromogenic detection.
Three washes for 5 min each time were performed in TTBS buffer and the membrane was incubated with horseradish peroxidase-conjugated anti-mouse (Santa Cruz Biotehnology) for 2 h at room temperature, diluted 1 2000 in 5% blocking reagent (Amersham) in TTBS buffer.
For amino-terminal sequencing of the midgut proteins, the proteins were electro-blotted to PVDF blot paper in a Xcell II Mini-Cell with transfer buffer (Invitrogen) and the membrane stained with 0.025% Coomassie Blue (R) in the absence of acetic acid.
Pellets were resuspended in an isotonic buffer and the membrane vesicles were isolated with spinning steps of 4000 g and 100.000 g.
