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. 2005 Aug 25;568(Pt 3):767–788. doi: 10.1113/jphysiol.2005.087858

Figure 2. Formation of a protein complex containing both DPP10 and KChIP3 requires Kv4.2 coexpression.

Figure 2

A, anti-Kv4.2, anti-KChIP3 and anti-DPP10 antibodies specifically detect their respective target proteins. The antibodies were checked by probing immunoblots with extracts from oocytes with or without Kv4.2, KChIP3 or DPP10 cRNA injection. B, anti-DPP10 and anti-HA antibodies detect the same HA/DPP10 proteins. In addition, the blots also show that neither antibody cross-reacts with Kv4.2 proteins. C, Kv4.2 proteins promote the coimmunoprecipitation of DPP10 and KChIP3. Oocytes expressing HA-tagged KChIP3 (HA/KChIP3) and DPP10 with and without Kv4.2 (as indicated) were treated with a reversible cross-linking reagent and solubilized. Goat (cntrl) IgG, anti-HA and anti-Kv4.2 antibodies were added to the extracts, and the immunoprecipitants were recovered by protein A/G Sepharose beads, denatured by SDS, and reduced by dithiothreitol. Immunoprecipitated proteins on Western blots were probed with anti-DPP10 (a), anti-KChIP3 (b), anti-Kv4.2, (c) and anti-HA antibodies (d). Samples of the starting extract (extract) are also provided for comparison. D, the coimmunoprecipitation of KChIP3 with DPP10 was made possible by the presence of Kv4.2 proteins. Oocytes expressing HA-tagged DPP10 (HA/DPP10) and KChIP3 with and without Kv4.2 were processed for immunoprecipitation in the same way as described earlier. Arrowheads point to the expected molecular masses of respective proteins, and arrows indicate the protein bands that show DPP10 and KChIP3 coimmunoprecipitate in the presence of Kv4.2, revealing the three-protein complex.