Ab103016

Rats went through procedures in Figure 1 without cannula placement, microinjection or locomotion test. Western blotting was performed as previously described (Zhang et al., 2018 (link)). Briefly, brain tissue containing the VLO was rapidly collected after animal anesthesia and decapitation. The tissue was dissected and homogenized in ice-cold lysis buffer. The homogenate was incubated on ice for 30 min and then centrifuged at 10,000 g for 15 min at 4°C. The supernatant was collected and stored at −80°C until use. Sample protein levels were measured using a bicinchoninic acid (BCA) protein assay. Loading proteins were separated on acrylamide gels and then transferred to pore size 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat milk, the membranes were probed for anti-rabbit 5-HT₆ receptor (Abcam, ab103016, 1:1,500, Cambridge, MA, United States), and anti-mouse actin (Sigma, A5316, 1:100,000, St Louis, MO, United States). Densitometry of each protein was evaluated by Image-J (NIH). Samples from each animal were run at least four times to minimize inter-blot variance. Raw values were normalized to actin.

Mice were anesthetized using pentobarbital sodium (50 mg/kg), and infused with saline, followed by 4% formaldehyde, from the base of the left ventricle. Brains were cut into slices of 40 μm thickness using a freezing microtome (Leica). Slices were washed with phosphate-buffered saline, and treated with 1% Triton-100, followed by goat serum, and overnight incubation with the primary antibody (rabbit anti-5-HT6R [1:1000; ab103016; Abcam, Cambridge, MA, USA] and mouse anti-GAD67 [1:500; MAB5406; Millipore]) at 4 °C. Slices were then incubated with the corresponding fluorophore-conjugated secondary antibody (Alexa Fluor 488 [1:500; A11034; Invitrogen], Alexa Fluor 594 [1:500; A11005; Invitrogen]) at room temperature for 1 h. The slices were then mounted using Fluoroshield mounting medium with 4′,6- diamidino-2-phenylindole (ab104139; Abcam). Images with fluorescence were captured by fluorescent microscopy (Nikon).

Brain tissues were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). Proteins were then separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using standard procedures [72 (link)]. The antibodies used were rabbit anti-HTR6 (1:500, Abcam #ab103016), rabbit anti-phospho-S6K (Thr389, 1:1,000, Cell Signaling Technology #9205), rabbit anti-S6K (1:1,000, Cell Signaling Technology #2708), rabbit anti-phospho-Akt (Ser473, 1:1,000, Cell Signaling Technology #9271), rabbit anti-Akt (1:1,000, Cell Signaling Technology #9272), rabbit anti-phospho-PKA (Thr197, 1:1,000, Cell Signaling Technology #4781), rabbit anti-PKA (1:1,000, Cell Signaling Technology #4782), rabbit anti-phospho-CREB (Ser133, 1:1,000, Millipore #06–519), rabbit anti-CREB (1:1,000, Millipore #AB3006), and mouse anti-α tubulin (1:10,000, GeneTex #GTX628802). Protein signals were visualized with horseradish peroxidase–conjugated secondary antibodies and ECL reagent (Thermo Fisher Scientific). Quantification of immunoblots was conducted with Image J software.