Syntaxin1a

Equal aliquots from each fraction separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 hr in 5% milk/Tris-buffered saline and probed overnight at 4°C with the following primary antibodies diluted in 5% milk/Tris-buffered saline containing 0.1% Tween-20: calnexin (Enzo Life Sciences, Farmingdale, NY, USA), syntaxin1a (Abcam, Cambridge, MA, USA), mGluR5, PKCε, phospho-(Ser729)-PKCε (all from Millipore, Billerica, MA, USA) and ERK1/2 and phospho-ERK1/2 (both from Cell Signaling Technology, Danvers, MA, USA). After incubation with an appropriate horseradish peroxidase-conjugated secondary antiserum (Jackson ImmunoResearch, West Grove, PA, USA), immunoreactive bands on the membranes were detected by ECL+ chemiluminescence reagents on an X-ray film (GE Healthcare, Piscataway, NJ, USA). Subsequently, blots were stripped and re-probed with calnexin and syntaxin-1a antibodies to monitor biotinylation of intracellular proteins and to normalize for unequal loading and/or transfer of proteins. The integrated band density of each protein sample was measured using NIH Image J software version 1.32j (RRID: SCR_003070). All primary antibodies used in this study are described in detail in Table 1.