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. 2003 Jan;14(1):156–172. doi: 10.1091/mbc.E02-02-0115

Figure 6.

Figure 6

Comparison of MLK3 properties with those of NIMA. (A) Homogenates made from HEK293 cells (Tot) were separated by centrifugation into a soluble (Sup) and insoluble fraction (Pel). The soluble fraction was incubated with either GST-Pin1 beads or GST-beads, which were then washed extensively before the addition of SDS sample buffer. Homogenate fractions as well as bead-bound material were subjected to immunoblot analysis using MLK3-specific antibodies. The bead-bound material shown (GST-Pin1 and GST) was derived from ∼10 times the volume of soluble extract shown in the Sup sample. (B) HEK293 cells were transfected with HA-tagged MLK3 (a and b) or NIMA (c and d) expression constructs for 9 h, fixed with methanol, and stained for HA (a and c) and γ-tubulin (b and d). The γ-tubulin–staining centrosomes are indicated by the closed arrowheads. (C) HEK293 cells were transfected with HA-tagged MLK3 (a–c) or NIMA (d–f) expression constructs for 33 h, fixed with methanol, and stained for DNA (a and d), HA (b and e), and phospho-histone H3 (c and f). Transfected cells are indicated by closed arrowheads, and untransfected prometaphase cells are indicated by open arrowheads. Scale bar, 10 μM.