Fp biotin

Competition experiments were performed in 50 mM phosphate buffer (pH 8) supplemented with 10 mM EDTA and 2 mM DTT. 75 nM bacterially expressed PREPL or immunopurified PREPL was pre-incubated with 10 μM inhibitor or DMSO (0.05% final concentration) as a control at 37°C for 15 min. This was followed by a reaction with 1.5 μM FP-biotin (Santa Cruz Biotechnology, sc-215056) at 37°C for 15 min. The reaction was stopped with the addition of SDS-PAGE loading buffer and denaturing the sample at 95°C for 10 min. Western blot analysis using a Streptavidin-HRP antibody (Agilent) was performed to evaluate the potential inhibition of PREPL. Membranes were stripped and relabeled with the GST antibody (Santa Cruz Biotechnology; sc138) to detect the presence of PREPL. Serine hydrolase inhibitors were kindly provided by Dr. Ben Cravatt (Scripps Research Institute, USA). Palmostatin B was purchased from Merck (cat. 178501). 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile was purchased from Vitas-M Laboratory (STK649251).