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. 2009 May 11;284(29):19361–19370. doi: 10.1074/jbc.M109.001644

FIGURE 3.

FIGURE 3.

DOR undergoes AIP4-dependent lysyl-ubiquitination. A, HEK293 cells stably expressing F-DOR or F-DOR-0cK were transfected with HA-tagged ubiquitin and, 24 h later, were stimulated with 10 μm DADLE for the indicated time period before extraction and immunoprecipitation under denaturing conditions with mouse anti-FLAG antibody (M2) and SDS-PAGE separation. Shown is a representative anti-HA (3F10)-HRP blot with untransfected HEK293 cells (no receptor expressed; 293), the F-DOR (left), and F-DOR-0cK (right). B, the same blot stripped and reprobed with anti-FLAG (M2)-HRP to show the relative size and amount of the major receptor species. C, HEK293 cells stably expressing F-DOR were transfected with HA-tagged ubiquitin and either empty pcDNA vector or mycAIP4-C/A construct and 24 h later were stimulated with 10 μm DADLE for 20 min before extraction and immunoprecipitation with mouse anti-FLAG antibody (M2) and SDS-PAGE separation. Shown is a representative anti-HA (3F10)-HRP blot. D, the same blots were stripped and reprobed with anti-FLAG (M2)-HRP to show the relative size and amount of the major receptor species. IP, immunoprecipitation; IB, immunoblot.