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. Author manuscript; available in PMC: 2006 Sep 28.
Published in final edited form as: J Biol Chem. 2002 Dec 23;278(10):8780–8785. doi: 10.1074/jbc.M208120200

Fig. 4. The effect of missense mutations of TBX5 on the interaction between TBX5 and NKX2.5 in vivo (A) and in vitro (B).

Fig. 4

A, HeLa cells were transiently transfected with expression plasmids for His-TBX5 and NKX2.5-HA. Total cell lysates containing His-tagged TBX5 and/or HA-tagged NKX2.5 were incubated with Ni-NTA beads, separated by 12% SDS-PAGE, and analyzed by Western blot with an anti-His antibody (upper panel) or with an anti-HA monoclonal antibody (middle panel). Proteins bound to Ni-NTA beads were washed with washing buffer, eluted, and fractionated by 12% SDS-PAGE and analyzed by Western blot with anti-mouse HA for NKX2.5. Lane 1, wild type (WT) TBX5 without co-transfection of NKX2.5; lane 2, wild type TBX5 with co-transfection of NKX2.5; lanes 2–9, TBX5 with Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I co-transfected with NKX2.5-HA, respectively. B, GST-NKX2.5 fusion protein and in vitro-translated His-tagged TBX5 were incubated with glutathione-Sepharose 4B beads, and proteins bound to the beads were washed with PBS, eluted, fractionated by 12% SDS-PAGE, and analyzed by Western blot with an anti-His antibody for detecting TBX5. Approximately equal amounts of wild type and various mutant TBX5 proteins were used in the GST pull-down assay as shown in the legend to Fig. 2B.

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