Zebrafish Arhgef11 is a homolog of human ARHGEF11 and is expressed during early embryogenesis. (A) Schematic depicting the percentage of identical (I) and similar (S) amino acids between known functional domains of zebrafish Arhgef11 and its human and fly homologs. An asterisk marks the domains omitted in the dominant-negative construct (ΔDHPH). (B) Ethidium bromide stained agarose gel showing RT-PCR products after amplification of the indicated fragments (blue arrows in A) at the listed developmental times, along with fragments amplified from `early' and `late' cloned constructs. Alternatively-spliced exons are indicated by pink bars in A. (C-E) WT embryos after whole-mount in situ hybridization using antisense probe for arhgef11 at the indicated developmental stages. Scale bar, 100 μm. (E') Cryosection through the indicated region in E. Abbreviations: nt, neural tube, no, notochord, pd, pronephric ducts. Scale bar, 50 μm. (F-I') Serum-starved cultured HEK293 cells expressing GFP, myc-Arhgef11, or myc-ΔDHPH. Cells in G were incubated with thrombin. F-actin is visualized by phalloidin in red (F-I), and Arhgef11 or ΔDHPH constructs are in green (H' and I', respectively) as detected by α-zRG. Scale bar, 20 μm. (J) Western blots using protein extracts from 6 hpf WT embryos injected as indicated. α-zRG detects both endogenous and overexpressed forms of full-length Arhgef11 (∼157 kDa) and overexpressed ΔDHPH (∼116 kDa), whereas &alpha-Myc only detects the overexpressed forms of each. (K) Schematics of chromosomal locations of zebrafish and human arhgef11 genes, showing synteny between the two chromosomes in the region encompassing arhgef11.
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