Fig. 1.

CLIM protects endogenous Lhx3 from proteasomal degradation in αT3 cells. (A) Cellular levels of Lhx3 and CLIM are regulated by the proteasome. Shown are Western blots of protein extracts that were treated for 6 h with increasing amounts of proteasome inhibitor lactacystin. (B) (Upper) CLIM deletion constructs consisting of CLIM2 full length (CLIM2FL; 1–373), dominant-negative CLIM (DN-CLIM; 245–341), and a C-terminal deletion (CLIMΔC; 1–277). DD, dimerization domain; LCCD, Ldb1/Chip conserved domain; NLS, nuclear localization signal; LID, LIM interaction domain. (Lower) Transfection of αT3 cells with Myc-tagged CLIM expression constructs. Cells are costained with specific Myc (red) and Lhx3 (green) antisera. Note the higher levels of endogenous Lhx3 in cells transfected with Myc-CLIM2FL and Myc-DN-CLIM. (C) (Left) Western blot of protein extracts of cells transfected with Myc-DN-CLIM or the empty vector as control. The same blot was probed with antisera against Lhx3, Myc and GAPDH. Note the higher levels of endogenous Lhx3 in cells transfected with Myc-DN-CLIM. (Right) No change in Lhx3 mRNA levels of the same transfected cells as measured by real-time RT-PCR (n = 3; values are mean ± SE). (D) The LIM domain mediates instability of LIM-HD proteins. mRNA encoding LIM-HD proteins Lhx3 and Isl1 with or without LIM domains were injected in one- to two-cell-stage zebrafish embryos. Embryos were fixed at 5 and 24 hpf and stained with a monoclonal Myc antibody. At 24 hpf, the focus is on trunk somites. Note that little or no Myc staining is detected in Myc-Lhx3 and Myc-Isl1-injected embryos, whereas ΔLIM proteins are readily detectable.