Figure 1.
Polytene squash preparations from male wild-type and JIL-1 null salivary glands labeled with H3S28ph antibodies.
(A) Wild-type and homozygous JIL-1z2/JIL-1z2 null (z2/z2) squash preparations labeled with H3S28ph (UP) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (B) Wild-type and JIL-1z2/JIL-1z2 null (z2/z2) squash preparations labeled with H3S28ph (CS) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (C) Polytene squash preparation from a JIL-1z2/JIL-1z2 null (z2/z2) salivary gland expressing a CFP-tagged JIL-1-CTD construct (JIL-1-CTD-CFP) labeled with H3S28ph (CS) antibody (in red); GFP/CFP antibody (in green), and Hoechst (DNA, in blue/gray). The male X chromosome is indicated by an X and the nucleolus by an n. Examples of interband labeling by the H3S28ph (CS) antibody are indicated by arrowheads in (B) and (C).
Figure 2.
Histone H3S10ph and H3S28ph antibody labelings of male salivary gland nuclei smush preparations.
(A) Double labeling with H3S10ph antibody (in red) and JIL-1 antibody (in green) demonstrating co-localization and the characteristic upregulation of JIL-1 and H3S10ph labeling on the male X chromosome (X). (B) Double labeling with H3S28ph (CS) antibody (in red) and JIL-1 antibody (in green). (C) Double labeling with H3S28ph (UP) antibody (in red) and JIL-1 antibody (in green). In contrast to the labeling of the H3S10ph antibody there was no discernable labeling above background of the autosomes or the male X chromosome by either of the two H3S28ph antibodies.
Figure 3.
Polytene chromosomes from wild-type and JIL-1 null salivary glands labeled with two different H3S28ph antibodies after heat shock treatment.
(A) Wild-type and homozygous JIL-1z2/JIL-1z2 null (z2/z2) squash preparations labeled with H3S28ph (CS) antibody (in red), Pol IIoser5 antibody (in green), and Hoechst (DNA, in blue/gray). There was no obvious labeling by the antibody above background of the heat shock puffs although they were robustly labeled by the Pol IIoser5 antibody. (B) Wild-type and homozygous JIL-1z2/JIL-1z2 null (z2/z2) squash preparations labeled with H3S28ph (UP) antibody (in red), Pol IIoser5 antibody (in green), and Hoechst (DNA, in blue/gray). This antibody weakly labeled heat shock puffs above background levels; however, such labeling was also observed in the JIL-1 null mutant background.
Figure 4.
JIL-1 does not co-localize with the paused form of RNA Polymerase II and is not upregulated at developmental puffs.
The polytene chromosome squash preparation from a wild type larvae was triple labeled with Pol IIoser5 antibody (in green), JIL-1 antibody (in red), and Hoechst (DNA, gray/blue). At many sites that showed especially high levels of Pol IIoser5 staining such as at developmental puffs, there was little or no JIL-1 antibody labeling. The male X chromosome is indicated by an X.
Figure 5.
H3S28 phosphorylation of mitotic chromosomes.
(A) S2 cell preparations labeled with H3S28ph (UP) antibody (in red), tubulin antibody (in green), and Hoechst (DNA in blue). The images show clear H3S28ph antibody labeling of chromosomes at pro- and metaphase, but little or no labeling of interphase nuclei. (B–C) Squash preparations from homozygous JIL-1z2/JIL-1z2 null (z2/z2) third instar larval brains labeled with H3S28ph (UP) or H3S28ph (CS) antibody (in red) and with Hoechst (DNA in blue). Mitotic chromosomes were robustly labeled by both antibodies.
Figure 6.
Immunoblot characterization of two different H3S28ph antibodies.
(A–D) Immunoblots of protein extracts from salivary glands (SG) or the CNS from wild-type (wt) or JIL-1z2/JIL-1z2 (z2/z2) larvae labeled with H3S28ph (UP) antibody (A), H3S28ph (CS) antibody (B), and H3S10ph antibody (C). Labeling with histone H3 (H3) antibody was used as a loading control (D) and as a marker for the relative migration of histone H3 (lane 6). The relative migration of molecular size markers is indicated in kD.
Figure 7.
14-3-3 antibody labeling of salivary gland chromosomes and nuclei.
(A–B) 14-3-3 antibody labeling of male polytene squash preparations before and after heat shock treatment. (A) Wild-type squash preparations labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (B) Wild-type squash preparations after heat shock treatment labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red), Pol IIoser5 antibody (in green), and Hoechst (DNA, in blue/gray). No or little specific labeling above background of either 14-3-3 antibody was discernable. (C–E) 14-3-3 localizes to the nuclear matrix surrounding the chromosomes. (C–D) Confocal sections of whole-mount salivary gland nuclei labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red) and Hoechst (DNA in blue). (E) Confocal section of a live salivary gland nuclei from a 14-3-3ε-GFP (in green) enhancer trap line co-expressing histone H2Av-RFP (in red).
Figure 8.
Immunoblot characterization of two different 14-3-3 antibodies.
(A) Immunoblot of protein extracts from salivary glands labeled with 14-3-3 (CS) or 14-3-3 (SZ) antibody. (B) Immunoblots of the relative distribution of 14-3-3 proteins in nuclear (NF) and cytoplasmic (CF) fractions (left panel) probed with the 14-3-3 (SZ) antibody. The quality of the fractionation was verified by labeling of the fractions with histone H1 antibody (middle panel) and tubulin antibody (right panel). The relative migration of the proteins based on the migration of molecular size markers is indicated in kD.
Figure 9.
Tethering of LacI-JIL-1 is not associated with upregulation of either H3S28ph or 14-3-3 at the LacO insertion site.
(A–C) Triple labelings with LacI antibody (in green), H3S10ph antibody (A) or H3S28ph antibody (UP) (B) or 14-3-3 (SZ) antibody (C) (in red), and Hoechst (DNA in blue/gray) of polytene squash preparations from larvae homozygous for the lacO repeat line P11.3. There is robust labeling by the H3S10ph antibody at the insertion site; however, there was no discernable signal above background levels when the preparations were labeld with either H3S28ph or 14-3-3 antibody.