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. Author manuscript; available in PMC: 2015 Feb 19.
Published in final edited form as: Biochem J. 2012 May 1;443(3):789–798. doi: 10.1042/BJ20112062

Figure 5. CK2 is required for Ihh/Wnt5a-induced Nkx3.2 protein degradation.

Figure 5

Figure 5

(A) Nkx3.2-Flag expression construct was co-transfected into ATDC5 cells with empty vector or SmoA1-HA expression vector in the absence or presence of HA-CK2α-KD and HA-CK2α′-KD expression plasmids. After 48 h of transfection, cells were harvested and total cell lysates were analyzed by western blotting.

(B) ATDC5 cells were transfected with Nkx3.2-Flag expression construct together with empty vector or Wnt5a expression vector in the absence or presence of HA-CK2α-KD and/or 3HA-CK2α′-KD expression plasmids for 48 h, and then total cell lysates were analyzed by western blotting.

(C) Nkx3.2-Flag expression vector was transfected into ATDC5 cells in the absence or presence of SmoA1-Flag expression plasmid. At 36 h post-transfection, cells were treated with DMSO, 100 μM DRB, or 100 μM TBB for an additional 12 h. Cells were then harvested and total cell lysates were analyzed by western blotting.

(D) ATDC5 cells were transfected with Nkx3.2-Flag expression plasmid in the absence or presence of Wnt5a expression vehicle for 48 h. DMSO, 100 μM DRB, or 100 μM TBB was included for the final 12 h prior to harvesting, and then total cell lysates were analyzed by western blotting.

(E) ATDC5 cultures were pre-treated with DMSO or 10 μM PMP for 24 h prior to addition of 100 μM DRB or 100 μM TBB. After an additional 12 h, cells were harvested and total cell lysates were analyzed by western blotting.

(F) Control or CK2α/β shRNA-harboring lentivirus was infected into ATDC5 cells. After 24 h, cells were treated with DMSO or 10 μM PMP for an additional 24 h, and then total cell lysates were analyzed by western blotting.

(G) Expression constructs for Nkx3.2-Flag and V5-Ubiquitin were transfected into 293T cells in the absence or presence of Myc-CK2α and Myc-CK2β expression plasmid for 24 h. Transfected cells were exposed to 20 μM MG132 for the final 6 h of transfection. Anti-Flag immunoprecipitates and total cell lysates were analyzed by western blotting.