Sc 5591

Samples were diluted in loading buffer (final concentration: 60 mM Tris, 10% (v/v) glycerol, 5% (v/v) mercaptoethanol, 2% (w/v) SDS, 0.025% (w/v) bromophenol blue, pH 6.8), incubated for 5 minutes at 95°C and submitted to gel electrophoresis using AnykD gels (Biorad, Hercules, CA), and blotted onto PVDF membranes (BioRad). Membranes were washed in TBS-T and blocked in TBS containing 5% (w/v) dry milk powder, which was also used for antibody incubations. Incubation in primary antibody against α4 (1:100, sc-5591, Santa Cruz Biotechnology, Heidelberg, Germany), α7 (1:1000, ab23832, Abcam, Cambridge, UK), or β2 (1:1,000, a gift from Dr. Cecilia Gotti, which we have characterized previously [23 (link)]) was performed overnight at 4°C on parafilm in a humidified container, followed by 3 × 10 minute washes in TBS-T and 1 hour incubation at 20–22°C in horseradish peroxidase-coupled secondary antibody (1:1,000, Dako, Glostrup, Denmark). After thorough washing in TBS-T, enhanced chemiluminescence Western blotting detection reagents (Western Lightning ECL Pro, Perkin Elmer, Waltham, MA) were used for signal detection and protein bands were visualized using a Chemidoc XRS system with Quantity One software (Biorad). Mean optical densities of bands were measured and their corresponding background measurement subtracted.

Total protein content was measured using a DC Protein Assay Kit (Biorad, Hercules, CA). Amounts of lysates containing equal protein content were then diluted in loading buffer (120 mM Tris, 20% (v/v) glycerol, 10% (v/v) mercaptoethanol, 4% (w/v) SDS, 0.05% (w/v) bromophenol blue, pH 6.8), incubated for 10 minutes at 95°C, submitted to gel electrophoresis in AnykD gel (Biorad), and blotted onto polyvinylidene fluoride membranes (BioRad). Membranes were washed in Tris-buffered saline with 0.1% Tween 20 (TBST) and blocked in TBS containing 5% (w/v) dry milk powder, which was also used for antibody incubations. Incubation with primary antisera directed against β2 (1:1,000, provided by Dr. Cecilia Gotti), α3, α4, α5, α6, 5-HT₃ (1:100 #sc-1771, sc-5591, sc-28795, sc-27292, sc-28958 Santa Cruz Biotechnology), α7, β4 (1:1,000 #ab23832 and 1:100 #ab156213 Abcam, Cambridge, UK) was performed overnight at 4°C on parafilm in a humidified container, followed by 3×10 minute washes in TBST and 1 h incubation at 21°C with horseradish peroxidase-conjugated secondary antibody (1:2,000, Dako, Glostrup, Denmark). After thorough washing in TBST, enhanced chemiluminescence Western blotting detection reagents (Western Lightning ECL Pro, Perkin Elmer, Waltham, MA) were used for signal detection and protein bands were visualized using a Chemidoc XR digital image analyser (Biorad).