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. 2015 Jan 9;290(9):5783–5796. doi: 10.1074/jbc.M114.628644

FIGURE 4.

FIGURE 4.

Interactions between PKA, ERM proteins, and DCC. PKA and DCC interact with all three ERM proteins. A and B, lysates prepared from IMR-32 cells in the absence or presence of 500 ng/ml netrin (A) or NG108-15 cells transfected with pRK5-DCC (B) were immunoprecipitated (IP) with control IgG or antibodies against ezrin (Ezr), DCC, or PKA RII subunits as indicated. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. C, left panel, whole cell lysate from differentiated, stage II-III hippocampal neurons (Hp) was separated by low percentage/high cross-linking SDS-PAGE and analyzed by immunoblotting with a pan-ERM antibody. The positions of ezrin (Ezr), radixin (Rxn), and moesin (Msn) are indicated. Right panel, whole cell lysates from differentiated NG108-15 cells (NG), IMR-32 cells (IMR), and hippocampal neurons (Hp) in triplicate were separated on a single, low percentage/high cross-linking SDS-PAGE gel and transferred to nitrocellulose. The membrane was cut into thirds, and each was analyzed by immunoblotting with antibodies specific for ezrin, radixin, and moesin. D, Triton-insoluble lysate from IMR-32 cells was incubated with cAMP-agarose in the absence or presence of 20 μm cAMP. After extensive washing, bead-bound proteins were eluted in sample buffer and analyzed alongside unfractionated whole cell extract (wce) by SDS-PAGE and immunoblotting as described in A. E, Triton-insoluble lysates from E18 rat brain were immunoprecipitated with non-immune IgG or anti-DCC antibody, and immunoprecipitates were analyzed by immunoblotting with antibodies against ezrin (Ezr), radixin (Rxn), and moesin (Msn) as indicated.