Anti calreticulin

Polyacrylamide gel electrophoresis was performed in an electrophoresis cell chamber (Bio-Rad, Hercules, CA, USA #1658004) at 70 V for the stacking gel; then, the voltage was changed to 120 V once the samples reached the separating gel. The proteins were then transferred via wet blotting onto polyvinyl difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA, #1620177) in a blotting cell (Bio-Rad, Hercules, CA, USA, #1703930) for 1 h at 4 °C with 135 V and 35 mA in blotting buffer (25 mM Tris, 192 mM glycine, 1% SDS, and 20% methanol). The membranes were blocked for 1–2 h with 5% milk powder in 1× TBS-T at RT and then incubated with either primary goat polyclonal antibodies (including goat polyclonal anti-mTrkB (1:5000; R&D Systems, Minneapolis, MN, USA, #AF1494), rabbit polyclonal anti-phosphoTrkB (Tyr816) (1:5000; Millipore, Burlington, MA, USA, #3587088), and rabbit anti-phospho TrkB (1:5000; Cell Signaling Technologies, Danvers, MA, USA, #46215)), chicken polyclonal anti-Calreticulin (1:2000; Thermofisher; #PA1902A), or goat-anti DARPP-32 (1:2000; R&D systems, Minneapolis, MN, USA, #AF6259) overnight at 4 °C on a rocker. Following this step, incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies was performed (with either anti-goat, anti-mouse, anti-chicken, or anti-rabbit antibodies (1:10,000)).