FIGURE 2.

Interaction of derlins with the ERAD substrates. A, 293T cells stably expressing SHH-HA precursor were transfected with FLAG-tagged DERL1, DERL2, or WASP plasmids as indicated. The cells were lysed in buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 1% Nonidet P-40 with protease inhibitors and immunoprecipitated (IP) by HA antibodies. Precipitates were separated on SDS-PAGE and immunoblotted (IB) with FLAG antibodies to detect the interaction with SHH. B is as in A, but with cells stably expressing N278A proteins. C is as in A but with cells stably expressing NHK.