goat anti-anillin  (Absolute Biotech Inc)

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: (A) Starting at the N terminus, the predicted domains of sea urchin anillin include a formin binding domain (FBD), a N-terminal domain (NTD), a myosin II binding domain (MBD), an actin binding domain (ABD), a candidate nuclear localization sequence (RTRRR), an anillin homology domain (AHD), and a plecstrin homology domain (PH). The dashed boxes for FBD, MBD, and ABD indicate approximate locations. (B) Immunoblot of affinity purified anti-sea urchin anillin PH domain antibody against the purified peptide antigen (Ag, lane 1) shows the expected 16 kDa immunoreactive band. Blotting this antibody against lysates of either unfertilized S . purpuratus eggs (UF, lane 2), or first cleavage embryos (CL, lane 3) reveals a ~120 kDa immunoreactive band. (C) Immunoblot of anti-human septin2 peptide antibody against LLC-PK1 cell lysate (lane 1 total protein left and immunobot right) and L . pictus first cleavage embryo lysate (lane 2 total protein left and immunoblot right) reveals a ~40–45 kDa immunoreactive species. (D-J) Anillin (green) staining of a S . purpuratus sperm aster stage early embryo (D-G) and adult coelomocytes (H-J) shows that anillin localizes to the nucleus and in early embryos to the cortical region. Embryos are co-labeled for microtubules (magenta) and DNA (blue), whereas coelomocytes are co-labeled for actin filaments (magenta) and DNA (blue). (K-M) Septin2 (green) staining of adult S . purpuratus coelomocytes also labeled for P-MyoRLC (magenta) and DNA (blue) demonstrates the expected staining of septin in stress fiber-like actomyosin bundles in phagocytes and in the flagella of vibratile cells (arrow in K).

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Binding Assay, Sequencing, Western Blot, Affinity Purification, Purification, Staining, Labeling

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: In dividing S . purpuratus embryos co-labeled for microtubules (magenta) and DNA (white; blue in L) in order to determine mitotic progression, anillin (green) staining is not obvious in late anaphase (A-C) but begins concentrating in the early cleavage furrow starting at the initiation of telophase (D-F) and continues through the midbody stage prior to abscission in late telophase (G-O). In off axis images the anillin staining appears as an entire ring similar to other CR markers (J-L). In L the phase contrast image is superimposed on the fluorescence image for context. The magnifications of A-O are equivalent and A-I plus M-O are confocal images whereas J-L are widefield images.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Labeling, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: Both septin2 (A-L, green) and anillin (M-T, green) mirror P-MyoRLC (A-T, magenta) staining in dividing embryos and begin as collections of clusters (A-D, M-P) that progress to tight rings (E-H, Q-T), and end concentrated in the midbody (I-L). L . pictus embryos appear in confocal images in A-L, S . purpuratus embryos appear in widefield images in M-T, and the magnifications of A-T are equivalent.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Staining

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: Septin2, and active myosin II (P-MyoRLC) localize together in the CR regions of isolated cortices (A-H) and progress from regularly spaced clusters in early stages (A-D) to dense assemblages of more patchy and linear structures in late stages (E-H). Anillin displays a similar co-distribution with active myosin II, as well as the analogous evolution from clusters in early cortices (I-L) to denser and more filamentous arrays in mid-late cortices (M-Q). Lower magnification images of myosin II (MyoHC) staining in cortices isolated early in cytokinesis (R) shows the presence of punctate clusters in a majority of cortices containing CRs (percentages graphed in T), whereas cortices isolated mid-late in cytokinesis (S) have a majority of cortices with patchy/filamentous CR patterns (percentages graphed in T). Bar in A = 10 μm and magnifications of A-Q are equivalent. Bar in R = 10 μm and magnification of R and S are equivalent. All cortices from S . purpuratus embryos.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Isolation, Staining

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: Septin2, active myosin II (P-MyoRLC) and F-actin staining all associate with clusters in early CRs (A-D), and with the denser, more linear arrays in late stage CRs (E-H). Anillin displays a similar association with active myosin II and F-actin in late stage CRs (I-L) and also codistributes with Rho A/B/C in the CR (M-P). Bar in A = 10 μm; magnifications of A-L are equivalent. All cortices from S . purpuratus embryos.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Staining

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: (A-F) Survey (A-C) and higher magnification views (D-F: enlarged white boxes in A-C) of isolated cortices from dividing embryos double labeled for P-MyoRLC (magenta in A-F) and either MyoHC (yellow in A, D), septin2 (green in B, E), or anillin (cyan in C, F). (G-L) The pairs of images that appear in G&J, H&K and I&L consist of a 10 μm x 3 μm XY image on the top paired with a corresponding 10 μm x 2 μm XZ image of the same clusters on the bottom. (M) In later stage CR regions of isolated cortices clusters become enlarged and appear to interact/coalesce with one another. Box and whisker plots (min/max with line at median) of small cluster spacing (N) and diameter (O) in early stage cortices stained by the three combinations of antibodies. Bar magnifications as indicated, with images in M equivalent in magnification to panel F. All cortices from S . purpuratus embryos.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Isolation, Labeling, Whisker Assay, Staining

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: (A-C) MyoHC (yellow) and P-MyoRLC (magenta) staining of early cytokinesis stage small clusters showing what appear to be mini-filaments arranged in chains (A) and rings (B, C). (D-G) Staining of P-MyoRLC (magenta) with either septin2 (green in D, E) or anillin (cyan in F, G) shows peripheral position of myosin II heads and the more central position of septin2 and anillin. (H-K) The central location of septin2 (green in H, I) and anillin (cyan in J, K) was confirmed by analyzing early small clusters with 2D line scans (H, J) and 3D surface plots (I, K) of relative staining intensities. Insets in H-K show images being analyzed and the line or area ROI–the images of clusters in I and K have been rotated to match the orientation of the 3D surface plots. (L) Box and whisker plots (min/max with line at median) of the percent of total early small clusters with centralized septin2 or anillin staining. Bar in A = 500 nm; magnifications of A-G are equivalent. All cortices from S . purpuratus embryos.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Staining, Whisker Assay

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: (A-D) MyoHC (yellow) and P-MyoRLC (magenta) staining of mature CR showing alignment of head-to-head minifilament chains (C, D). (E-L) Septin2 (green) and P-MyoRLC (magenta) labeling of a late stage CR region showing the network-like structure of septin2 filaments in close association with myosin II (H, L) using both 3D-SIM (E-H) and STED (I-L) imaging. (M-U) Anillin (cyan) and P-MyoRLC (magenta) staining of a mature CR indicates that anillin is more punctate in distribution and in close proximity to myosin II (M-P). The cortex in Q-V is highly contracted (Q shows a low magnification confocal view) and in this CR remnant the STED imaging of anillin appears similar to a network. White boxes in C, G, K, O, T correspond to the regions that appear at higher magnification in the insets labeled D, H, L, P, U. Bars = 10 μm. All cortices from S . purpuratus embryos.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Staining, Labeling, Imaging

Journal: PLoS ONE

Article Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

doi: 10.1371/journal.pone.0252845

Figure Lengend Snippet: (A-C) Whole embryo (EMB) treated with LatB and stained for septin2 (green), P-MyoRLC (magenta), and DNA (blue) and viewed using a through focus projection of a confocal Z series. The embryo contains a circumferential band of clusters containing myosin II and septin2 and the DNA staining indicates that it has undergone nuclear division during mitosis but not cytokinesis. (D-F) Cortex (CTX) isolated from a LatB treated embryo shows that septin (green) and P-MyoRLC (magenta) localize to a concentrated stripe of clusters. (G-I) Whole embryo (EMB) treated with LatA and stained for anillin (cyan) and P-MyoRLC (magenta) shows they colocalize in a stripe in an embryo which has undergone karyokinesis (I, DNA in blue). (J-L) Cortex (CTX) isolated from a LatA-treated embryo demonstrating a band of anillin and P-MyoRLC clusters. Bar = 10 μm in A; magnifications of A-L are equivalent. L . pictus embryos in A-F, S . purpuratus embryos in G-L.

Article Snippet: For anillin immunoblots HRP conjugated secondary antibodies were used and detected by chemiluminescence using a Immun-Star HRP kit, with imaging performed on a ChemiDoc XRS molecular imaging system from Bio-Rad.

Techniques: Staining, Isolation

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Midbody assembly appears normal in KIF20B-depleted cells. (A, B) PRC1 localizes to the central spindle in anaphase in both siLUC and siKIF cells. In early and late midbodies (MB), PRC1 lines the microtubules of the midbody flanks and extends into the cell on the microtubule network. It also forms two distinct disks around the core of the midbody (white arrow). (C, D) MKLP1/KIF23 localizes to the central spindle in anaphase in siLUC and siKIF cells. In early and late midbodies, it is found in the center of the midbody (dark zone) in control or KIF20B depleted cells. By contrast, Aurora kinase B (AURKB) localizes to the flanks of the midbody in early and late stages in both siLUC and siKIF cells. (E, F) Phospho-T232-Aurora B (pAURKB), representing “activated” Aurora B kinase, is diffusely localized during anaphase in both siLUC and siKIF cells. In early midbodies, pAURKB localizes to the center dark zone and inner flanks as a diffuse blob, but appears as a more compact disk in later midbodies, similar in siLUC and siKIF cells. (G, H) Anillin (ANLN) localizes to the furrowing cell cortex during anaphase in siLUC and siKIF cells. In the early midbody stage, ANLN forms a wide ring around the center of the midbody. In late midbodies, ANLN can be found in the center of the midbody as well as at the constriction sites. These localizations were not disrupted in KIF20B-depleted cells. (I, J) α-Actinin-4 (ACTN4) is distributed on the entire cell cortex but clearly accumulates in the cleavage furrow during anaphase in both siLUC and siKIF cells. In early midbody stage, ACTN4 enriches in a half-circle shape at the edges of both daughter cells underneath the midbody. In the late midbody stage, there is no longer enrichment of ACTN4 around the midbody, in both siLUC and siKIF cells. Images in panels A and B and E–J were taken with a DeltaVision deconvolution microscope and those in C and D with a wide-field microscope. At least 15 midbody-stage and five anaphase cells imaged for each marker and condition. Scale bars are 5 µm for all images.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Microscopy, Marker

Journal: Molecular Biology of the Cell

Article Title: Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

doi: 10.1091/mbc.E17-08-0495

Figure Lengend Snippet: Pattern of anillin and VPS4 recruitment is consistent with late-stage maturation defect in KIF20B-depleted midbodies. (A) Left, example deconvolution images of endogenous VPS4 and anillin localization in representative midbodies of HeLa cells. Right, schematic representations of staining in images on left. Arrows point to the central bulge region, and arrowheads point to constriction sites. White, anillin; green, VPS4; red, tubulin. Scale bar, 1 µm. (B) The percentage of midbodies with anillin enriched in the center is increased, and the percentage with VPS4 enriched in the center is decreased. n = 106 siLUC-treated cells, and 109 siKIF-treated cells from two coverslips each of two independent experiments. (C) The percentage of constriction sites (cs) that have anillin enrichment was increased, while the percentage of constriction sites that have VPS4 enrichment was decreased, in KIF20B depleted midbodies, but did not reach statistical significance. n = 48 siLUC constriction sites in 38 midbodies, and n = 46 siKIF constriction sites in 34 midbodies. (D) Bar plot of anillin and VPS4 co-occurrence in midbodies shows KIF20B-depleted midbodies are significantly shifted out of the latest-stage category (VPS4-only) and into the early (anillin-only) and transitional (anillin plus VPS4) categories. n = 106 siLUC-treated midbodies, n = 109 siKIF-treated midbodies. (E) Detailed schematic representations and raw tallies of subcategories of anillin and VPS4 enrichment data plotted in bar graphs in B–D. White space in microtubules symbolizes the central dark zone, and pointed ends symbolize constriction sites (cs). Anillin (blue) or VPS4 (green) enrichment was scored at midbody centers or constriction sites. * p < 0.05; **** p < 0.0001; n.s., not significant (Fisher’s test for B and C, Chi-square test for D).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal DM1α (α-tubulin; 1:500) was from Abcam; rat anti-TUBA1A (clone YL ½; 1:750) was from Novus Biologicals; mouse polyclonal anti-CEP55 (1:200) was from Abnova; mouse anti-Aurora kinase B (AURKB; 1:300) was from BD Biosciences; rabbit anti-phospho-T232-Aurora kinase B (pAURKB; 1:200) was from Rockland; rabbit anti-KIF20A (A300-879A; 1:100) was from Bethyl Labs; goat anti-anillin (ANLN; 1:300), mouse monoclonal anti-ANLN (1:100), rabbit anti-MKLP1 (sc-867; 1:100), rabbit anti-PRC1 (1:50), mouse monoclonal anti-human-spastin (3G11/1; 1:50), and mouse anti-human-MPP1(KIF20B; 1:300) were from Santa Cruz; rabbit anti-mouse-Kif20b (1:500) was custom-made by Covance ( Janisch, Vock, et al. , 2013 ); rabbit anti-cleaved-caspase 3 (CC3; 1:200) and rabbit anti-phosphohistone H3 (PH3, Alexa Fluor 647 conjugated; 1:400) were from Cell Signaling; rabbit anti-α-ACTININ4 (ACTN4; 1:250) was from Millipore; and rabbit anti-VPS4 (1:500) was from Sigma Aldrich.

Techniques: Staining

Journal: Molecular Biology of the Cell

Article Title: Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission

doi: 10.1091/mbc.E15-02-0101

Figure Lengend Snippet: Defects in formation of the contractile ring in augmin-depleted cells. (A–D) Immunostaining for RhoA (A), myosin IIA heavy chain (B), and anillin (D) or staining of F-actin by rhodamine-phalloidin (C) in RNAi-treated cells. MTs were visualized by either immunostaining (B, D) or EGFP–α-tubulin (A, C). (E) Left, for quantification in F and G, the dividing cells were categorized based on the progression of furrow ingression, which was quantified using a furrowing index, defined as (1 – furrow width/cell length). See Materials and Methods for details. Middle and right, example of an ROI used for quantification in F (a line profile across the division plane, green) and G (a line profile along the cortex, magenta). The light blue box indicates the cytoplasmic region used for background subtraction. (F) Normalized intensity of RhoA, myosin IIA, F-actin, or anillin staining at the cleavage furrow during the progression of furrowing. Each data point corresponds to the average of two peak intensities of a line profile (E) from each single cell. Data points were binned at a 0.2 interval of the furrowing index, and the mean ± SE of the bins is shown as squares with a line. Data were normalized to the mean intensity of controls at the bin of the furrowing index 0.6–0.8. At least 24 cells (RhoA), ≥28 cells (myosin IIA), ≥32 cells (F-actin), or ≥24 cells (anillin) from at least two independent experiments per condition were analyzed. Depletion of Aug6 significantly decreased anillin accumulation at the cleavage furrow (* p < 0.05, ** p < 0.01, *** p < 0.001; t test). (G) Normalized intensities of RhoA, myosin IIA, F-actin, and anillin along the cell cortex. Data from the cells with a furrowing index of 0.4–0.6 (corresponding to the furrowing phase). Two line profiles were derived from both sides of each single cell. Mean ± SE of ≥8 cells (RhoA), ≥6 cells (myosin IIA), ≥13 cells (F-actin), or ≥9 cells (anillin) from at least two independent experiments per condition. Scale bars, 5 μm.

Article Snippet: Rat monoclonal anti–α-tubulin antibody (YOL1/34; AbD Serotec, Raleigh, NC), mouse monoclonal anti-aurora B antibody (611082; BD Biosciences, Franklin Lakes, NJ), rabbit monoclonal anti-PRC1 antibody (ab51248; Abcam, Cambridge, United Kingdom), goat polyclonal anti-PRC1 antibodies (sc-9342; Santa Cruz Biotechnology, Dallas, TX), mouse monoclonal anti-NEDD1 antibody (H00121441-M05; Abnova, Taipei, Taiwan), goat polyclonal anti-anillin antibodies (sc-54859; Santa Cruz Biotechnology), rabbit polyclonal anti-ECT2 antibody (sc-1005; Santa Cruz Biotechnology), goat polyclonal anti–citron kinase (sc-1848; Santa Cruz Biotechnology), mouse monoclonal anti-RhoA antibody (sc-418; Santa Cruz Biotechnology), and rat monoclonal anti-GFP (GF090R; Nacalai Tesque, Kyoto, Japan) were purchased from suppliers as indicated.

Techniques: Immunostaining, Staining, Derivative Assay

Journal: Molecular Biology of the Cell

Article Title: Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission

doi: 10.1091/mbc.E15-02-0101

Figure Lengend Snippet: Exogenous expression of EGFP-anillin rescues early cytokinesis defects in Aug6-depleted cells. (A) Immunostaining for anillin in RNAi-treated cells expressing EGFP–α-tubulin or anillin-EGFP. Scale bar, 5 μm. (B) Normalized intensity of anillin staining at the cleavage furrow. Mean ± SE of ≥5 cells from two independent experiments per condition. Asterisks indicate statistically significant differences ( p < 0.05, t test). An example of the ROIs used for quantification is shown in Supplemental Figure S3G. (C, D) Time plot of furrow-width change (C) and maximum furrowing rate (D) in RNAi-treated EGFP–α-tubulin– or anillin-EGFP–expressing cells. Mean ± SE of ≥6 cells from two independent experiments per condition. p values from t tests. (E) Frequencies of cytokinesis defects in control or Aug6-depleted cells. At least 17 cells from four independent experiments per condition were analyzed. In anillin-EGFP–expressing cells, the early regression induced by Aug6 depletion was dramatically rescued, whereas it was not rescued in other control cells (EGFP–α-tubulin– or Cep55-EGFP–expressing cells).

Article Snippet: Rat monoclonal anti–α-tubulin antibody (YOL1/34; AbD Serotec, Raleigh, NC), mouse monoclonal anti-aurora B antibody (611082; BD Biosciences, Franklin Lakes, NJ), rabbit monoclonal anti-PRC1 antibody (ab51248; Abcam, Cambridge, United Kingdom), goat polyclonal anti-PRC1 antibodies (sc-9342; Santa Cruz Biotechnology, Dallas, TX), mouse monoclonal anti-NEDD1 antibody (H00121441-M05; Abnova, Taipei, Taiwan), goat polyclonal anti-anillin antibodies (sc-54859; Santa Cruz Biotechnology), rabbit polyclonal anti-ECT2 antibody (sc-1005; Santa Cruz Biotechnology), goat polyclonal anti–citron kinase (sc-1848; Santa Cruz Biotechnology), mouse monoclonal anti-RhoA antibody (sc-418; Santa Cruz Biotechnology), and rat monoclonal anti-GFP (GF090R; Nacalai Tesque, Kyoto, Japan) were purchased from suppliers as indicated.

Techniques: Expressing, Immunostaining, Staining, Control