Fig. 7.

Effects of I74 on the nuclear localization, DNA-binding activity, and structure integrity of Myb2. (A) A conserved isoleucine, I74, in 4HA-Myb2 was mutated to alanine (I74A) or proline (I74P). Subcellular localization of 4HA-Myb2, I74A, and I74P was examined by IFA with a mouse anti-HA antibody. The signal was shown as green fluorescence (FITC). The nucleus was stained with DAPI, and cell morphology was recorded by phase-contrast microscopy. Bar, 5 μm. Two distinct forms of I74A are indicated as I74A-1 and I74A-2. (B) Total lysates (TL) from cells overexpressing 4HA-Myb2, I74A, and I74P were fractionated into the nuclear (N) and cytosolic (C) fractions. Protein samples were separated in 12% gel for Western blotting with a rat anti-HA antibody (right panel). Duplicate blots were examined by the anti-acetyl-histone H3(Lys9) (α-H3K9-Ac) and anti-CypA1 (α-CypA1) antibodies. (C) I74, in the recombinant protein rMyb2(48∼148) (lane 2) was mutated to alanine or proline to produce rI74A(48∼148) (lane 3) or rI74P(48∼148) (lane 4), respectively. EMSA was performed using coincubation of the recombinant proteins with a γ-³²P-labeled MRE-1 probe (lane 1). The reaction mixtures were separated in 12% gels. The signal was detected by autoradiogram. (D) The secondary structures of the recombinant proteins rMyb2(48∼148), rI74A(48∼148), and rI74P(48∼148) were monitored by far-UV CD spectra. E. The ternary folding of rMyb2(48∼148), rI74A(48∼148), and rI74P(48∼148) was examined by fluorescence spectroscopy. All fluorescence emission spectra were measured at 300 to 400 nm and 25°C. Protein samples were dissolved in PBS buffer (pH 7.4) with 1 mM DTT. rMyb2(48∼148) was also denatured with 6 M guanidine hydrochloride.