Anti gfp antibody

Cell pellets were resuspended in PBS at an OD₆₀₀ = 2. Samples were sonicated and centrifuged at maximum speed for 20 min at 4 °C. The soluble, insoluble and total fractions were resolved on 12% w/v SDS-PAGE gel and transferred onto a PVDF membrane. Immunodetection was performed using anti-GFP antibody (BD Biosciences) and membranes were developed with ECL Chemiluminescent HRP Substrate (Millipore) according to the manufacturer’s protocols. Densitometry bands analysis was performed using the Image J software.

Cell lysates were prepared in 50 mM Tris-HCl at pH 7.4, 50 mM NaCl, 0.5% Triton X-100, phosphatase and protease inhibitors, centrifuged at 16,000 × g for 20 min. Protein concentration was quantified using Bradford assay (Pierce). In total, 500 μg of protein from cell lysate were incubated overnight at 4 °C with 10 μg/ml of anti-ENDOA2 (Santa Cruz) or anti-GFP antibodies (BD Pharmingen), and finally incubated with protein A/G magnetic beads (88802, Thermo Scientific) for 2 h at 4 °C. The immunocomplexes were washed three times in lysis buffer and resuspended in 2X Laemmli’s sample buffer. For western-blot analysis, 50 μg of protein was loaded for each condition.

HEK 293 cells were co-transfected with C-terminal YFP-fused and N-terminal Xpress-tagged H₃Rs. Forty-eight hours after transfection, cells were washed with ice-cold PBS and lysed in buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, and 0.5% Triton X-100 plus protease cocktail inhibitor on ice for 10 min. The lysates were then centrifuged at 10,000× g for 10 min. The supernatants were incubated with protein A-sepharose (GE Healthcare, Chalfont St. Giles, UK) and anti-GFP antibodies (BD Bioscience Clontech, San Jose, CA, USA) overnight at 4 °C. The beads were washed five times with lysis buffer and resuspended in 4-fold concentrated Laemmli buffer (200 mM Tris-HCl pH 6.8, 4% SDS, 40% glycerol, 0.02% bromophenol, βME 0.5 M). Non-specific background was determined by the transfection of N-terminal Xpress-tagged H₃Rs without H₃R-YFP.