Methods for Identification of cGKI Substrates

Abstract

The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different methods inter alia co-immunoprecipitation (Co-IP) and cGMP-agarose affinity purification. In the following chapter, we will describe the preparation of cell or tissue lysates, the procedures of cGMP-agarose affinity purification and co-immunoprecipitation, and finally the separation and analysis of the protein complexes by SDS-PAGE or mass spectrometry.

Regulation of the Na+‐K+‐2Cl− cotransporter by cGMP/cGMP‐dependent protein kinase I after furosemide administration

“…β‐actin was used as loading control. The separated proteins were electroblotted onto a poly(vinylidene difluoride)‐membrane (Immobilon, Millipore GmbH, Schwalbach, Germany) using a semi‐dry transfer system, as described previously . Proteins were detected with primary antibodies (see above) followed by incubation with secondary antibodies coupled to horseradish peroxidase (HRP), including mouse IgG HRP‐conjugated (dilution 1 : 20 000) (Sigma Aldrich, Taufkirchen, Germany) and rabbit IgG HRP‐conjugated (dilution 1 : 50 000) (Dianova, Hamburg, Germany).…”

Interaction of cCMP with the cGK, cAK and MAPK Kinases in Murine Tissues

“…The samples were pretreated as described in the sections above. Proteins were separated by 11.5% sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; equal volumes (15 μL, concentration as indicated in figure legends) of protein were loaded in every lane) and transferred to a PVDF membrane using a semi-dry transfer system (also described in [ 18 ]). For one hour, the blotted membrane was blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 150 mM NaCl, pH 8.0, with 0.1% Tween 20), and then washed with TBST.…”