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Fig. 4. Tethering of LacI-JIL-1 `kinase dead' to lacO repeat insertion lines. (A) Double labeling of a polytene squash preparation homozygous for the lacO repeat line P11.3 with LacI antibody (in green) and with Hoechst 33258 (in blue and gray). The arrow indicates chromatin structure perturbations at the insersion site. Arrowheads indicate chromatin structure perturbations and ectopic contacts at other sites. (B) Triple labeling of a polytene squash preparation homozygous for the lacO repeat line P11.3 with LacI antibody (in green), with H3S10ph antibody (in red) and with Hoechst 33258 (in blue and gray). The upper panel shows LacI-JIL-1 tethering compared to LacI-JIL-1 kinase dead tethering in the lower panel. Arrows indicate the approximate lacO repeat insertion sites. (C) Triple labeling of a polytene squash preparation heterozygous for the lacO repeat lines P11.3 and 4D5 with LacI antibody (in green), with JIL-1 antibody (in red) and with Hoechst 33258 (in blue and gray). Arrows indicate the approximate lacO repeat insertion sites. (D) Levels of H3S10 phosphorylation were reduced when LacI-JIL-1 kinase dead was expressed, compared with wild-type levels and when LacI-JIL-1 was expressed in homozygous lacO P11.3 lines. Immunoblots were performed on extracts from third instar larval salivary glands and labeled with JIL-1, H3S10ph, histone H3 and tubulin antibodies. The upper arrow indicates LacI-JIL-1 or LacI-JIL-1-kd, whereas the lower arrow shows the location of wild-type JIL-1.

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