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. 2016 Mar 31;7(17):23056–23071. doi: 10.18632/oncotarget.8521

Figure 5. BMAL1 and RORα do not bind to HIF-1α in NP cells.

Figure 5

A. Immunoprecipitation (IP) of BMAL1 and CLOCK from NP cells cultured under normoxia or hypoxia for 24 h followed by Western blot analysis using anti-HIF-1α, anti-BMAL1 and anti-CLOCK antibodies. BMAL1 bound to CLOCK, but neither BMAL1 nor CLOCK bound to HIF-1α irrespective of oxygen tension. Preimmune rabbit IgG was used as a negative control for IP assays. B. Pulldown of HIF-1α did not show co-precipitation of BMAL1, CLOCK, or RORα. HC: heavy chain of IgG, nsp: non-specific (C) Pulldown of HIF-1α showed co- precipitation of HIF-1β/ARNT, association was higher under hypoxia. D., E. Treatment of NP cells with RORα inhibitor, ML-176 (10 μM), showed no effect on nuclear levels of HIF-1α. Densitometric analysis shown in (E) was performed on blots from 3 independent experiments, Data is represented as mean ± SE, * p < 0.05.