P0704

Cells were lysed in RIPA buffer, and the cell lysates were treated with PNGase F (peptide N-glycosidase F) for 3 h at 37 °C according to the manufacturer’s instructions (#P0704, New England Biolabs). The reaction was stopped by the addition of 5 × loading buffer followed by western blot analysis using anti-NRP1 antibody (#3725, Cell Signaling Technology).

Y receptor protein levels in SkM were determined by immunoblotting. Protein lysates were subjected to deglycosylation under denaturing reaction conditions according to manufacturer’s instructions (P0704, New England Biolabs). Following deglycosylation, lysates were heated at 60°C and cooled to room temperature. 20 μg of protein was loaded in 10% SDS gels and transferred to PVDF membranes using Stain-Free technology (Bio-Rad). Membranes were incubated in primary antibodies Y1r, Y2r, and Y5r (NBP1-59008, NB100-56480, NB100-1538, respectively, Novus Biologicals) diluted (1:1000) in a chemiluminescent blocking buffer (blØk^TM – CH, Millipore) overnight at 4°C. After the overnight incubation, membranes were washed with TBST before incubation with appropriate secondary antibody (anti-rabbit, Proteintech or anti-goat, Thermo Scientific) at a 1:25000 dilution in blocking buffer at room temperature. Membranes were visualized after incubation in SuperSignal^TM West Femto (Thermo Scientific) on the Bio-Rad ChemiDoc MP.