Fig 2. MTG16 and HIF1a co-precipitate.

A. COS-7 cells were transfected with MTG16 and HIF1α and examined by IP-Western as described in Materials and Methods using α-MTG and α-HIF1α antibodies. MTG16 (precipitated with a-MTG) co-precipitated HIF1α (top panel) and the reciprocal experiment showed HIF1α to co-precipitate MTG16 (lower panel). The input of HIF1α in 3% whole cell lysate and the corresponding input of MTG16 are shown in upper and lower panel, respectively. Control experiments with α-β-actin showed lack of non-specific co-precipitation of HIF1α or MTG16 (lanes 4). The same membrane was used for the immunoblotting in upper and lower panel. B. Similar experiments as in A were carried out on Raji-MTG16 cells, which were induced for 12 h with 20 ng/ml doxycycline (dox) to express MTG16 and exposed to 4% O₂ to induce HIF1α. MTG16 (precipitated with α-MTG) co-precipitated HIF1α (top panel) and the reciprocal experiment showed HIF1α to co-precipitate MTG16 (lower panel). The input of HIF1α or MTG16 in 3% of whole cell lysate is shown in lanes 2. As negative control, cells under exposure to 20% O₂ not incubated with doxycycline showed no reaction with α-HIF1α or α-MTG (lanes 4). One out of at least three experiments performed is shown in A and B.