Journal: eLife

Article Title: Arid1a restrains Kras-dependent changes in acinar cell identity

doi: 10.7554/eLife.35216

Figure Lengend Snippet:

Article Snippet: Antibody , GFP antibody (rabbit monoclonal) , Cell Signaling Technology , Cell Signaling Technology Cat# 2956P, RRID: AB_10828931 , .

Techniques: TaqMan Copy Number Assay, Software, Plasmid Preparation

Journal: PLoS ONE

Article Title: The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

doi: 10.1371/journal.pone.0111905

Figure Lengend Snippet: ( A, B ) Kis1-GFP was visualized with Sid4-CFP (marks SPB; A) and Mis6-2mRFP (marks kinetochore; B). I: interphase cells; M: mitotic cells. Cell shapes are outlined. ( C ) Kymograph of Kis1-GFP and Cnp3-tdTomato in a cell filmed from G2 phase to anaphase. Kis1/Mis19 dispersed at the onset of SPB separation (filled arrowhead) and reappeared after chromosome segregation (open arrowhead). The length of the downward arrow corresponds to 2 min. ( D ) The amount of Kis1-GFP during the cell cycle. The cdc25-22 mutant was used to synchronize the cell cycle at G2. Cells were shifted to 36°C for 4 h and then back to 25°C. Samples were then taken every 20 min (0–120 min). A sample for asynchronous cells (AS) was taken before the temperature shift. Top: Immunoblotting was performed with anti-GFP and anti-α-tubulin (control). Bottom: Kis1-GFP level was normalized with that of α-tubulin (red). Populations of cells with Kis1-GFP dots (green), binucleate cells (blue), and septated cells (black) are also shown. ( E ) Kis1-GFP was visualized with Sid4-CFP and Mis6-2mRFP in the cdc2-as (analog-sensitive) mutant. The ATP analog 1NM-PP1 was added to cells to inhibit the Cdc2 kinase activity, and images were acquired after 5 min after drug addition (analog, +). A mock-treated cell (analog, −) and cdc2 + cells are also shown. Cells were cultured at 25°C except in (D). Scale bars, 5 µm.

Article Snippet: For ChIP samples, 500 µl extract was incubated with or without anti-GFP Living Colors Full-length polyclonal antibody (1∶250; Clontech) for 1.5 h. Protein G–coupled DynaBeads (Life Technologies) were then added and incubated for 1.5 h. Beads were washed twice with Buffer I, once with Buffer I containing 0.5 M NaCl, once with Buffer II (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and twice with TE.

Techniques: Mutagenesis, Western Blot, Activity Assay, Cell Culture

Journal: PLoS ONE

Article Title: The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

doi: 10.1371/journal.pone.0111905

Figure Lengend Snippet: ( A ) Cells coexpressing Mis6-2GFP and Cnp3-tdTomato were imaged. ( B,C ) ChIP assays. Percentages of DNA precipitates of the centromeric region ( cnt and imr ) and the arm region ( lys1 ) were measured as scaled to total input DNA using quantitative PCR. Black bars (+), immunoprecipitation with anti-GFP; gray bars (−), mock-treated negative control. Error bars indicate standard deviation ( n = 3 reactions) ( B ) ChIP of Mis6-2GFP in WT or in kis1-1 cells grown at 36°C for 6 h. ( C ) ChIP of Kis1-GFP. ( D ) Localization of Kis1-GFP in WT and mis6-302 cells grown at 36°C for 6 h. Percentages indicate the proportion of interphase cells in which Kis1-GFP colocalized with both Cnp3-tdTomato and Sid4-CFP. ( E ) Ten-fold serial dilution assay. WT or kis1-1 cells expressing either GFP (−) or Mis6-GFP ( mis6 ) were grown on EMM plates at 25, 30, or 32°C. ( F ) A proposed model for localization dependency of Kis1/Mis19, Mis6 and Cnp1/CENP-A at kinetochores. ( G ) (a,b) Spindle phenotypes of mis6-302 cells. Time-lapse imaging of GFP-Atb2, Nup40-mCherry, and Sid4-CFP in mis6-302 was done at 36°C (6–9 h). (a) Blob spindles; (b) spindles with the dim midzone; (c) frequencies of spindle defects in WT, kis1-1 , and mis6-302 cells. Scale bars, 5 µm.

Article Snippet: For ChIP samples, 500 µl extract was incubated with or without anti-GFP Living Colors Full-length polyclonal antibody (1∶250; Clontech) for 1.5 h. Protein G–coupled DynaBeads (Life Technologies) were then added and incubated for 1.5 h. Beads were washed twice with Buffer I, once with Buffer I containing 0.5 M NaCl, once with Buffer II (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and twice with TE.

Techniques: Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Standard Deviation, Serial Dilution Assay, Expressing, Imaging

Journal: PLoS ONE

Article Title: The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

doi: 10.1371/journal.pone.0111905

Figure Lengend Snippet: ( A ) Genetic crossing of kis1-1 and the mis16-GFP-kan or mis18-GFP-kan mutant was performed, and spores were germinated on YE5S plates at 25°C. Colonies were then replica-plated onto YE5S plates with phloxine B (PB) or with kanamycin (G418) and incubated at 36°C or 25°C, respectively. The temperature-sensitive colonies (arrowheads) were sensitive to G418 without exception, indicating that the double mutant was inviable. mis16 and mis18 were found on the chromosome III, whereas kis1/mis19 was on chromosome II, excluding the possibility of genetic linkage between kis1/mis19 and mis16/mis18 . ( B ) WT or kis1-1 cells expressing Mis18-GFP from plasmids were observed after growth at 25 or 36°C for 6 h. Percentages of cells without Mis18-GFP dots are shown (n≥100). ( C ) Localization of Kis1-GFP in WT, mis16-53 , or mis18-262 cells grown at 25 or 36°C for 6 h. Frequencies are also given (n≥100). Scale bars, 5 µm. ( D ) Overexpression of Mis16-GFP suppressed the temperature sensitivity of kis1-1 . Cells harboring plasmid pREP1-mis16-GFP ( mis16 OP ) were spotted on EMM plates and incubated at 25 or 32°C. Cells harboring pREP1-GFP were spotted as controls. ( E ) Coimmunoprecipitation (IP) of Kis1 and the Mis16–Mis18 complex. Cell extracts (input) were prepared from cells expressing (+) or not expressing (−) the indicated proteins. Kis1-myc (left) and Mis18-GFP (right) were immunoprecipitated with anti-Myc or anti-GFP, respectively. IP samples as well as 2.5% of the input were analyzed.

Article Snippet: For ChIP samples, 500 µl extract was incubated with or without anti-GFP Living Colors Full-length polyclonal antibody (1∶250; Clontech) for 1.5 h. Protein G–coupled DynaBeads (Life Technologies) were then added and incubated for 1.5 h. Beads were washed twice with Buffer I, once with Buffer I containing 0.5 M NaCl, once with Buffer II (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and twice with TE.

Techniques: Mutagenesis, Incubation, Expressing, Over Expression, Plasmid Preparation, Immunoprecipitation

Journal: bioRxiv

Article Title: Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

doi: 10.1101/163279

Figure Lengend Snippet: a) Pictures of Malpighian tubules with esg ts -driven expression of GFP (control) and indicated transgenes. Quantification of esg+ cells is given on each picture (see also Supplementary Figure 3f). (b) Drawing of the DIAP1 locus. Exons are represented in cyan, the DIAP1-4.3 enhancer in dark green and the insertion site of the DIAP1-LacZ reporter (J5C8) is in red. Regions bound in ChIP-seq (MACS peaks) by Svb and Yki are indicated in green and magenta,respectively. (c) Svb co-immuno-precipitates with Yki in S2 cells. Svb::GFP and Yki::HA, or a mutated form of Yki substituting the WW domains (YkiWW::HA), were expressed in S2 cells. Protein immuno-precipitated by anti-GFP were blotted with anti-GFP and anti-HA antibodies. (d) esg ts was used to drive the expression of yki , svb REP ( ovoA ), and yki together with svb REP in RNSCs. The expression of DIAP1 was followed by the activity of DIAP1-lacZ . (e) Expression of the DIAP1-4.3-GFP enhancer was followed by immuno-staining against GFP (green) and Hindsight (Hnt, red) revealing the antagonistic influence of Yki and OvoA on RNSCs.

Article Snippet: Immuno-precipitated samples were separated by SDS-PAGE and transferred to PVDF membranes, then blotted using anti-GFP (TP401, Acris Antibodies, 1:10000) and anti-HA (Covance, 1:2.000) antibodies.

Techniques: Expressing, ChIP-sequencing, Activity Assay, Immunostaining