Extraction and Quantification of Membrane Proteins

Whole cell lysates were extracted in ice-cold RIPA lysis buffer, sonicated, kept on ice for 30 minutes, and centrifuged with 14000 g for 30 minutes at 4°C. PMFs were extracted using a membrane protein extraction Kit (Sango Biotech, China), in accordance with the manufacturer’s instruction. Detailed steps are shown in S1 File. Protein concentration was determined by bicinchoninic acid assay using bovine serum albumin as a standard. Equal amounts of protein (60 μg for whole cell lysates and 40 μg for PMFs) were subjected to immunoblotting. The densitometry values of whole cell lysates were normalized by the expression of GAPDH. The primary antibodies are mouse polyclonal anti-D₅R (Santa Cruz, USA) and rabbit polyclonal anti-CCK_BR (NOVUS, USA) whose specificities have been reported [29 (link),30 (link)].