Figure 8.

PKM2 and HIF-1 do not interact in NP cells. A) Western blot analysis after immunoprecipitation of HIF-1α shows that HIF-1α does not interact with PKM2 in rat NP cells. Image is representative of 3 independent experiments. B) Western blot analysis after immunoprecipitation of PKM2 shows no evidence of PKM2-HIF-1α interaction in NP cells. Image is representative of 3 independent experiments. C) Western blot analysis of HIF-1α, PKM2, and PHD3 using cytoplasmic and nuclear fractions of NP cells. Images are representative of 3 independent experiments. D) Schematic of WT, demethylase deficient (H321A), and truncated (ΔN80, lacks PKM2 binding capability) JMJD5 constructs used in panels E–G. E, F) HIF activity in NP cells after overexpression of WT (E) or mutant (F) JMJD5 constructs (n = 3). G) HIF activity in chondrocytes was also unaffected after overexpression of WT or mutant JMJD5 (n = 4). H) Measurement of HRE reporter activity in PHD3-silenced NP cells after pretreatment with PKM2 stabilizers (n = 3). Luciferase assays were performed with 3 technical replicates per experiment. Data are means ± sem. *P < 0.05.