Glp 1r

The expression of proteins extracted from the primary neurons and the right cerebral cortex of rats was analysed by Western blot. The protein samples were separated on a 12% sodium dodecyl sulfate (SDS)/polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with GLP-1R (Abcom, UK), phosphorylated AKT, ERK, p38, JNK and total AKT, ERK, p38, JNK, Bcl-2, Bax, Bcl-xl, Bad, cleaved caspase-3, -8, and -9, cleaved PRAP and GAPDH antibodies (Cell Signaling Technology, USA) at 4 °C overnight. Immunoreactive bands were visualized by increased chemiluminescence (Millipore, USA) using corresponding horseradish peroxidase-conjugated IgG secondary antibodies (Cell Signaling Technology, USA). The images were captured by the gel imager (UVITEC, UK) and quantified using Quantity One software (Bio-Rad, USA).

Primary neurons were fixed in 4% paraformaldehyde for 30 min at room temperature. After incubation in 1% BSA, 5% goat serum and 0.2% Triton X-100 in PBS for 2 h at 4 °C, GLP-1R (Abcom, UK) and MAP2 antibodies (Millipore, USA) from different species were sequentially applied overnight at 4 °C and corresponding secondary antibodies were applied for 1 h at room temperature, followed by counterstaining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI).