Figure 5.

AIP Protects BCL6 from FBXO11-Mediated Proteasomal Degradation

(A) HEK293T cells were transfected with either a FLAG-tagged FBXO11 or empty vector (EV-FLAG), together with MYC-tagged AIP. A majority of the whole-cell extracts (WCEs) were subjected to immunoprecipitation (IP) using FLAG antibody (rows 1 and 2), and the rest of the WCEs were used in immunoblotting (rows 3 and 4).

(B) HEK293T cells were transfected with either FLAG-tagged BCL6 or EV-FLAG, together MYC-tagged AIP (Leontiou et al., 2008). A majority of the WCEs were subjected to IP using FLAG antibody (rows 1 and 2), the and the rest of the WCEs were used in immunoblotting (rows 3 and 4).

(C) OCI-LY7 DLBCL cells were lysed and immunoprecipitated with antibodies to IgG, AIP, UCHL1 and immunoblottedfor AIP and UCHL1.

(D) OCI-LY7 DLBCL cells were lysed and immunoprecipitated with antibodies to IgG, BCL6, UCHL1 and immunoblotted for BCL6 and UCHL1.

(E) OCI-LY7 DLBCL cells were lysed and immunoprecipitated with antibodies to IgG, FBXO11 and BCL6 and immunoblotted for BCL6 and FBXO11. β-actin was used as a loading control.

(F) HEK293T cells were transfected with MYC-tagged AIP or EV-MYC, FLAG-tagged FBXO11, HIS-tagged BCL6, and hemagglutinin (HA)-tagged ubiquitin. Where indicated, cells were treated with MG132 post-transfection for 2.5 h to inhibit proteasomal degradation.

(G) HEK cells were transfected as in (F) and FLAG-tagged UCHL1. Cells were harvested and subjected to IP BCL6. Corresponding WCEs are shown.

(H) OC1-LY7 cells were stained with AIP, BCL6, UCHL1, and FBXO11. DAPI was used as a nuclear stain. Arrowheads show areas of co-localization.