Rabbit anti ha

Western blot was performed to measure the total levels of 5-HT1ARs, Rab proteins, clathrin, myosin V, phospho-dynamin I, and dynamin I in vehicle and ethanol-treated cells. Cells were lysed with a solubilization buffer containing protease and phosphatase inhibitor cocktails (MilliporeSigma cat#PPC1010). Equal amounts of proteins were loaded in 10% or 12% SDS-PAGE gels for gel electrophoresis and then transferred to nitrocellulose membranes. The following primary antibodies were used: rabbit anti-HA (Novus Biologicals cat# NB600–363), mouse anti-Rab4A (Santa-Cruz cat# sc-312), mouse anti-Rab5 (Santa-Cruz cat#sc-46692), rabbit anti-Rab11 (Santa-Cruz cat# sc-9020), mouse anti-clathrin heavy chain (Santa-Cruz cat#sc-12734), mouse anti-myosin Va (Santa-Cruz cat# sc-365986), mouse anti-phospho-dynamin-I (DHSB cat#3D3), and rabbit anti-dynamin-I (Invitrogen cat#pa1–660). The secondary antibodies including goat anti-mouse IgG-HRP (Invitrogen cat# G-21040) and goat anti-rabbit IgG-HRP (Cell Signaling cat# cat#7074S) were applied accordingly. The band intensity was quantified using FIJI software and the relative intensity of each band was normalized to that of GAPDH. Data are presented as relative to the vehicle treatment for each condition.

Immunofluorescence, immunoprecipitation, and western blot assays were performed as previously described. For western blots, anti-epitope tag primary antibodies were used at 1/1000. For immunofluorescence assays, primary antibodies were used at 1/500: mouse anti-HSV-1 ICP0 (Santa Cruz); rabbit anti-HA (Novus Biologicals); mouse anti-FLAG (Novus Biological); mouse anti-GFP (Santa Cruz); mouse anti-Myc-c (Novus Biologicals); rabbit anti-GPCMV IE2. Secondary antibodies anti-rabbit or anti-mouse IgG HRP-linked secondary antibodies for western blot (Cell Signaling) were used at 1/5000 and anti-mouse/rabbit IgG FITC or TRITC were used at 1/1000 for immunofluorescent staining. High titer recombinant defective adenovirus expressing GFP (AdGFP) was generated by Welgen Inc..

The effect of prolonged ethanol exposure on constitutive recycling of 5-HT1ARs was determined as described previously (Luessen et al. 2016 (link)). Surface 5-HT1ARs were labeled with rabbit anti-HA (Novus Biologicals cat#NB600–363) for 60 min at 4°C, followed by warming up cells to 37°C to induce receptor internalization. Any remaining, non-internalized antibody-bound 5-HT1ARs on the surface were blocked at 4°C using non-fluorescent secondary antibody goat anti-rabbit IgG-HRP (Cell Signaling Technology cat#7074P2). Cells were again warmed up to 37°C to allow internalized 5-HT1ARs to recycle back to the surface in 5, 15, or 30 min. The recycled 5-HT1ARs on the surface were probed with goat anti-rabbit Alexa Fluor 488 (Invitrogen cat#A32731TR). After fixation and permeabilization, internal (non-recycled) 5-HT1ARs were labeled using goat anti-rabbit Alexa Fluor 594 (Invitrogen cat#A-11012). Similar to internalization, the amount of recycling was calculated as a ratio of the intensity of Alexa Fluor 488 over that of Alexa Fluor 488 and 594 combined (recycled 5-HT1ARs/total surface 5-HT1ARs before assay) at an indicated time. Data are presented relative to basal 5-HT1AR levels for each treatment condition.