Figure 3. Fbxo22 mediates the heme-induced degradation of Bach1.

(A)HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged proteins. Twenty-four hours post-transfection, cells were treated with MG132 for 3 hours, collected for immunoprecipitation (IP) and immunoblotting. WCE, whole-cell extract.

(B)KP and KPK cells were treated with MLN4924 for 3 hours, collected for IP with either nonspecific IgG or with an antibody against Bach1, treated with hemin where indicated, and immunoblotted as indicated. l.ex., long exposure; s.ex., short exposure.

(C)A549 cells infected with lentiviruses expressing 2 different shRNAs targeting Fbxo22 (shFbxo22) under the control of a dox-inducible promoter were treated (where indicated) with dox for 24 hours. Cells were then treated with either CHX or hemin, collected at the indicated times, lysed, and immunoblotted as indicated. * denotes a nonspecific band.

(D)Two different KP-sgTom and KP-sgFbxo22 clones were treated with hemin, collected at the indicated times, lysed, and immunoblotted as indicated.

(E)HEK293T cells were transfected with either an EV or the indicated FLAG-tagged constructs. Twenty-four hours post-transfection, cells were treated with MLN4924 for 3 hours before collection for IP and immunoblotting. WCE, whole-cell extract; l.ex., long exposure; s.ex., short exposure.

(F) H2009 cells infected with lentiviruses expressing either a dox-inducible FLAG-tagged WT Bach1 or Bach1(Y11F) were transfected with either an EV or HA-tagged Fbxo22. Cells were treated with dox for 24 hours. Dox was then washed out and, after 4 hours, cells were treated with CHX, collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ±SEM. l.ex., long exposure; s.ex., short exposure.