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. Author manuscript; available in PMC: 2012 May 27.
Published in final edited form as: Cell. 2011 May 27;145(5):732–744. doi: 10.1016/j.cell.2011.03.054

Figure 2. PKM2 Interacts with HIF-1α.

Figure 2

(A) GST pull-down assays were performed with GST or GST-HIF-1α (531–826) and lysate prepared from HEK293 cells grown in media containing light isotopes (12C6-14N2-Lys and 12C6-14N4-Arg) or heavy isotopes (13C6-15N2-Lys and 13C6-15N4-Arg), respectively. The precipitated proteins were analyzed by Q-TOF mass spectrometry (Luo et al., 2010). The mass spectrum of a tryptic PKM2 peptide is shown. Monoisotopic peaks derived from PKM2 bound to GST-HIF-1α (531–826) are 4 Da greater in their mass/charge ratio (m/z) and have higher relative intensity compared to the corresponding peaks due to non-specific binding of PKM2 to GST.

(B) Co-IP was performed in HeLa cells exposed to 1% O2 for 24 h.

(C) Co-IP was performed using nuclear lysates (NL) prepared from HeLa cells transfected with PKM2-V5 vector and exposed to 1% O2 for 24 h.

(D and E) GST pull-down assays were performed with GST or GST fusion protein containing the indicated amino acid residues of HIF-1α (upper panels) and whole cell lysates (WCLs) from HeLa cells expressing PKM2-V5. bHLH, basic helix-loop-helix; PAS, Per-ARNT-Sim; TAD, transactivation domain; ID, inhibitory domain.