Prpt6

Following synaptosomal preparation, protein levels were normalized and loaded onto a 7.5% SDS/PAGE gel and then to a membrane using a transfer apparatus (Bio-Rad, Richmond, CA, USA). Membranes were incubated in blocking buffer for 1 h before being incubated in pRpt6 (ProSci, 1:850, Poway, CA, USA), total Rpt6 (Abcam, 1:1000, Cambridge, MA, USA), or βactin (Cell Signaling, 1:1000, Beverly, MA, USA) primary solutions overnight at 4 °C. Then, membranes were incubated in the appropriate secondary (Cell Signaling, 1:20,000) antibody for either four hours at 4 °C (pRpt6 and tRpt6) or one hour at room temperature (βactin) and prepped in a chemiluminescence solution for 3 min. Images were captured and densitometry was performed using NIH Genesys. The pRpt6 antibody was generated commercially (ProSci) against a synthetic peptide (NH2-CALRND(pS)YTLHK-OH) as described previously [16 (link)]. Western blotting was conducted in the same regions as IF images were taken except as follows. Tissue punches from both the anterior and posterior RSC were fractionated together into one sample and from the entire basolateral amygdala complex rather than just the lateral amygdala. Then, 15 μg of protein was loaded into each well per animal per ROI for blotting.