Tethering of LacI-JIL-1 is not associated with upregulation of either H3S28ph or 14-3-3 at the <i>LacO</i> insertion site.

<p>(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 <i>lacO</i> 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.</p

Histone H3S10ph and H3S28ph antibody labelings of male salivary gland nuclei smush preparations.

<p>(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.</p

14-3-3 antibody labeling of salivary gland chromosomes and nuclei.

<p>(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 IIo^ser5 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).</p

Polytene chromosomes from wild-type and <i>JIL-1</i> null salivary glands labeled with two different H3S28ph antibodies after heat shock treatment.

<p>(A) Wild-type and homozygous <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (CS) antibody (in red), Pol IIo^ser5 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 IIo^ser5 antibody. (B) Wild-type and homozygous <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (UP) antibody (in red), Pol IIo^ser5 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 <i>JIL-1</i> null mutant background.</p

Immunoblot characterization of two different H3S28ph antibodies.

<p>(A–D) Immunoblots of protein extracts from salivary glands (SG) or the CNS from wild-type (wt) or <i>JIL-1^z2/JIL-1^z2</i> (<i>z2/z2</i>) 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.</p

Polytene squash preparations from male wild-type and <b><i>JIL-1</i></b><b> null salivary glands labeled with H3S28ph antibodies.</b>

<p>(A) Wild-type and homozygous <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (UP) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (B) Wild-type and <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) 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 <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) 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).</p

Binding affinity of Chromator-CTD for microtubules determined by spindown assays.

<p>(A) Immunoblot analysis of spindown assays of Chro-CTD binding to various concentrations of taxol-stabilized microtubules (MT). 10 µg of Chro-CTD (lane 1) was incubated with microtubules at concentrations ranging from 0.125–2.5 µM (lane 2–9). Chro-CTD was detected with mAb 6H11 and microtubules with tubulin antibody. (B) Binding curve for Chro-CTD with microtubules based on three independent experiments indicated by squares, circles, and triangles, respectively. The calculated Kd was 0.23 µM.</p

Chromator and tubulin immunoprecipitation and pulldown assays.

<p>(A) Immunoprecipitation of lysate from S2 cells using tubulin antibody and detected with Chromator antibody. Chromator is detected in the tubulin ip (lane 3) and in the S2 cell lysate (lane 1) but not in the GST antibody control ip (lane 2). (B) Immunoprecipitation of lysate from S2 cells using Chromator antibody and detected with tubulin antibody. Tubulin is detected in the Chromator ip (lane 3) and in the S2 cell lysate (lane 1) but not in the beads only control (lane 2). (C) A full-length Chromator GST-fusion construct (Chro-FL-GST) pulls down tubulin from S2 cell lysate as detected by tubulin antibody (lane 3). A GST-only pull down control was negative (lane 2). Lane 1 shows the position of tubulin in the S2 cell lysate.</p

Spindown assay mapping of the Chromator interaction domain with tubulin.

<p>(A) Diagram of Chromator indicating the domains to which GST-fusion proteins were made for mapping. (B–C) In the spindown experiments the Chromator GST-fusion protein constructs were incubated with taxol stabilized microtubules. Assembled microtubules and associated proteins were then pelleted by centrifugation at 75,000 rpm for 20 min. For immunoblot analysis the pellet (B) and supernatant (C) were separated, fractionated by SDS-PAGE, immunoblotted and probed with anti-GST and anti-tubulin antibody. In these experiments Chro-FL, Chro-CTD, and Chro-M were detected in the pellet fraction (B) whereas Chro-NTD and Chro-421 were detected in the supernatant (C). This confirmed the Chro-M domain as sufficient for mediating interactions with tubulin. Tubulin for all five experimental conditions were only detectable in the pellet (B). (D) Immunoblot of the respective GST fusion proteins used in the spindown assays labeled with a GST mAb. The relative migration of molecular weight markers is indicated to the right of the immunoblots in kDa.</p

A Chro-M subdomain is minimally required for polymerized tubulin binding activity.

<p>(A) Diagram of Chromator indicating the domains to which GST-fusion proteins were made for mapping including the overlapping set of Chro-M1-M3 spanning the Chro-M domain. (B) Pulldown assays with purified bovine tubulin incubated with Chromator-GST fusion proteins under conditions where microtubules were assembled and stabilized by taxol. Bound proteins were washed, fractionated by SDS-PAGE, immunoblotted, and analyzed using a tubulin specific antibody. Whereas Chro-421, GST and Chro-M1-M2 showed no pull-down activity (lane 3–6), Chro-CTD, Chro-M, and Chro-M3 were all able to pull-down tubulin (lane 1, 2, and 7). (C) Pulldown assays with bovine TRITC-labeled tubulin incubated with Chromator-GST fusion proteins under conditions where microtubules were prevented from forming by colchicine. Bound proteins were washed, fractionated by SDS-PAGE, immunoblotted, and analyzed for TRITC fluorescence. Whereas Chro-NTD, Chro-421, and Chro-M1-M3 showed no pull-down activity (lane 2, 4, 6, 7, and 8), Chro-FL, Chro-CTD and Chro-M were all able to pull-down tubulin (lane 1, 3, and 5). (D) Immunoblot of the respective GST fusion proteins used in the pulldown assays labeled with a GST mAb. The relative migration of molecular weight markers is indicated to the right of the immunoblots in kDa.</p