Fig. 5: PFKFB4 hinders the ubiquitylation of HIF-1α by binding to the E3 ubiquitin ligase FBXO28.

A FBXO28-Halo and HIF-1α were overexpressed in HEK293T cells, and an FBXO28 pulldown was performed using Halo resin. FBXO28 and associated proteins were eluted by TEV-cleavage. Immunoblot was performed with FBXO28 and HIF-1α antibodies. B Upper panel: HIF-1α IP from lysate of NCH421k cells stably expressing HA-ubiquitin, in the presence of the proteasome inhibitor MG132 (500 nM, 6 hours) and with and without FBXO28 silencing (3 days). The immunoblot (IB) shows HA-ubiquitin and HIF-1α levels. The bracket indicate poly-ubiquitinated HIF-1α and the arrow shows the expected migration of unmodified HIF-1α (also applies to 5D and 5E). Lower panel: HIF-1α and FBXO28 protein levels in the input lysates used in the IP above. β-actin is displayed as a loading control in all panels. C Scheme of the HIF-1α protein domains including the ubiquitinylated lysines which were mutated to alanine (K532, K538, K547) (Adapted from ref. [54]). D Upper panel: HIF-1α IP from lysate of NCH421k cells stably expressing HA-ubiquitin and silenced (3 days) for endogenous HIF1A, in the presence of MG132 (500 nM, 6 hours), with and without PFKFB4 silencing and overexpression of wild-type or lysine-mutated HIF-1α. The IB shows HA-ubiquitin and HIF-1α levels. Lower panel: PFKFB4 protein levels in the input lysates used in the IP above. E Upper panel: HIF-1α immunoprecipitated (IP) from lysate of HEK293T cells in the absence and presence of MG132 (500 nM, 6 hours) and PFKFB4 overexpression (3 days). HIF-1α was overexpressed in all conditions. The IB shows ubiquitin (Ub) and HIF-1α levels. Lower panel: PFKFB4 protein levels in the input lysates used for the IP above.