Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Characterization of the molecular interactions between Cav1.3, SK3, and HCN channels using co-immunoprecipitation and immunoblot. Proteins were membrane-extracted in CL47a solubilization buffer (IP1) or in CL48 buffer (IP2) and immunoprecipitated using anti-Cav1.3 ( A ), anti-SK3 ( B ), and anti-Kv4.3 antibodies ( C ). Blots were performed on starting material, P2 membrane fraction (P2), or on solubilized P2 proteins (sol) or after immunoprecipitation (IP1, IP2). Blots were probed with anti-SK3 (( A ), right panel), anti-Cav1.3 (( B ), right panel), anti-HCN4 (( A , B ) left panels; ( C ), right panel), anti-Kv4.3 (( C ), left panel), and anti-HCN2 (( C ), middle panel). P2: enriched P2 membrane fraction. IP1: immunoprecipitated proteins in CL47a buffer. IP2: immunoprecipitated proteins in CL48 buffer. sol: solubilisate. WB: Western blot (immunoblot).

Article Snippet: Optimization of extraction and purification conditions were individually searched for the six channel protein complexes testing eight solubilization buffers of varying stringencies from Logopharm (GmbH, March, Germany).

Techniques: Immunoprecipitation, Western Blot, Membrane