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. Author manuscript; available in PMC: 2019 Aug 10.
Published in final edited form as: Cancer Lett. 2018 May 5;429:1–10. doi: 10.1016/j.canlet.2018.04.041

Figure 5. The enzymatic activity of ASPH is involved in the protein-protein interaction between RB1 and CDK complexes.

Figure 5

(A) IB results of CDK2, CDK4, and RB1 were determined in the IP products of HEK-293T treated as indicated. CDK2, CDK4, and ASPH were measured in the WCL. (B) IB results of cyclin D1, cyclin E, and RB1 were determined in HEK-293T cells treated as indicated. GAPDH, cyclin D1, cyclin E, and RB1 were measured in the WCL. (C) HA-RB1 and CDK4 were determined in the IP products of HEK-293T cells transfected with HA-RB1 in combination with EV, myc-ASPH, or myc-ASPH variant 3 (Myc-ASPHv3) which does not contain the enzymatic domain of ASPH. Myc tag, CDK4, and tubulin were measured in the WCL. The WCL was collected from HEK-293T cells 48 hours post sub-culture. (D) ASPH, HA-RB1, and CDK4 were analyzed in the IP products of HEK-293T cells transfected with HA-RB1 in combination with myc-EV, myc-ASPH, or myc-ASPHH675R which only has 20 % of enzymatic activity. The WCL was collected 48 hours post transfection. (E) The effects of DFO, DMOG, and specific ASPH inhibitor, MO-I-1151 on the protein interaction between CDK4 and RB1. HA-RB1 and CDK4 were measured in the IP products of HEK-293T transduced with myc-EV or myc-ASPH. HA-RB1 was transiently transfected in the HEK-293T-EV or HEK-293T-ASPH for 48 hours, and the treated HEK-293T cells were sub-cultured. 48 hours later, the WCL was collected from the treated HEK-293T cells which were challenged with different concentrations of DMOG, DFO, and MO-I-1151 24 hours before harvesting.