L 35s methionine

The pcDNA3 plasmids containing wild type or mutant GIRK cDNA (1 μg) were incubated with the TnT coupled Reticulocyte Lysate System (50 μL, Promega) and L-[³⁵S]-Methionine (>1,000 Ci/mmol at 10 mCi/ml) (Amersham Biosciences) for 90 min at 30 °C. This system utilizes crude reticulocyte lysate which could contain microsomes, and has been widely used to synthesize membrane proteins in vitro in a cell-free system^{62 (link)}. Translated proteins were subjected to in vitro cleavage reaction at 37 °C for 2 h with 0.2 μg caspase-1 (Calbiochem) or caspase-3 (BD Biosciences) in a buffer containing (in mM): 20 PIPES, 100 NaCl, 10 DTT, 1 EDTA, 0.1% CHAPS, and 10% sucrose. Products were separated by electrophoresis and visualized by exposure to autoradiographic BioMax MS films (Kodak).

[1-¹⁴C] oleic acid, L-[methyl-³H] carnitine, L-[³⁵S] methionine, and enhanced chemiluminescence detection reagents were bought from Amersham Pharmacia Biotech (Piscataway, NJ, USA), the BCA reagent was from Pierce (Rockford, IL, USA). The ABI DyeDeoxy Terminator Cycle Sequencing Kit was obtained from PE-Applied Biosystems (Foster City, CA, USA).