Fig. 2.

Enrichment and purification of the FLAG-tagged GC-A receptor from stably expressing HEK293 cells. (A) Cell fractionation and western blot analyses demonstrated that FLAG-tagged GC-A is predominantly localized in the plasma membrane of HEK293 cells. ERK1/2 and PMCA were used as markers for the cytosolic and membrane fractions, respectively. GC-A was detected with anti-GC-A serum and anti-FLAG IgG. (B) Western blot analysis demonstrated that immunoprecipitation of FLAG-tagged GC-A from the cell membrane fraction led to a 13-fold enrichment of the protein (2 μg protein per lane). (C) The silver-stained gel illustrates the step-wise purification of the receptor (10 μg protein per lane). (D) The immunoprecipitated GC-A protein was separated by SDS/PAGE. After Coomassie staining, the protein band at 130 kDa was excised and subjected to in-gel digestion with trypsin.