Ro 20 1724

The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441). All secondary antibodies for Western blots were obtained from LI-COR, and for immunofluorescence Alexa-488, −594, and −633 conjugated secondary antibodies were obtained from Invitrogen. Forskolin was obtained from Sigma; apamin was obtained from Millipore; Ro 20–1724 was purchased from Santa Cruz, and KT5720, CNQX, and D-AP5 were purchased from Tocris.

cAMP production was assessed using a Homogeneous Time-Resolved Fluorescence (HTRF) cAMP G_i kit (Cisbio, Codolet, France). In short, cAMP-cryptate, a donor, is added to lysed cells to compete with intracellular cAMP to bind a monoclonal-cAMP-d2 antibody, which acts as an acceptor. In case cAMP-cryptate binds to the antibody, the donor transfers energy to the acceptor, commonly called FRET, and a fluorescent signal is detected with a lag time. 6 × 10⁴ cells HEK 293T cells were seeded in 384-well Greiner low volume plates, coated with Poly-D-Lysine (100 µg/mL, Merck Millipore, Burlington, VT, USA) and transfected as explained above. Forty-eight hours after seeding, cells were incubated with 1x stimulation buffer, including 100 µM of Ro20-1724 (Santa Cruz Biotechnology, Dallas, TX, USA), a phosphodiesterase inhibitor, and GM1 or vehicle. After 2 h, cells were stimulated with 5 µM forskolin and incubated for 45 min. cAMP-crypate and monoclonal anti-cAMP-d2 were added, and the TRF signal was measured on Spark 10 M at 320/620 nm and 320/655 nm, respectively. cAMP levels were determined by standard curve interpretation. Data are presented as percentage of forskolin-stimulated eGFP-transfected cells at baseline. On each plate, transfection with D2R was included and stimulated with 10 µM of quinpirole in order to have a positive control.