anillin anti rabbit  (Santa Cruz Biotechnology)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cell size and polarization determine cytokinesis furrow ingression dynamics in mouse embryos

doi: 10.1073/pnas.2119381119

Figure Lengend Snippet: Apical polarity disrupts the recruitment of furrowing regulators independently of cell adhesion. ( A and C ) Representative live and immunofluorescence images of circumferentially dividing blastomeres undergoing the 16–32 cell division from embryos previously injected with control (example shown in A has 19 cells and in C has 16 cells) or PARD6B shRNA (example shown in A has 16 cells and in C has 16 cells). Note that both Anillin ( A ) and p-Myosin ( C ) are underrepresented at the side of contractile ring that overlaps with the apical domain in controls, and this bias is abolished upon PARD6B depletion (yellow arrows). Also note that the apical domain is intact during division in controls (green arrows). ( B ) Apical:basal fluorescence ratio of Anillin in outer blastomeres undergoing the 16–32 cell division from embryos previously injected with control ( n = 9 blastomeres from nine embryos) or PARD6B shRNA ( n = 9 blastomeres from nine embryos; unpaired two-tailed t test, **** P < 0.0001). ( D ) Apical:basal fluorescence ratio of p-Myosin in outer blastomeres undergoing the 16–32 cell division from embryos previously injected with control ( n = 9 blastomeres from nine embryos) or PARD6B shRNA ( n = 10 blastomeres from 10 embryos; unpaired two-tailed t test, **** P < 0.0001). ( E ) Representative live and immunofluorescence images of a circumferentially dividing blastomere undergoing the 16–32 cell division from a H2B:RFP-expressing embryo cultured in Ca +2 -free media, in which the furrow overlaps with the apical domain (example has 16 cells). Note that Anillin was underrepresented at the apical side (yellow arrow) and the apical domain is intact during division (green arrow). ( F ) Apical:basal fluorescence ratio of Anillin in outer blastomeres undergoing the 16–32 cell division from embryos cultured in Ca +2 -free media ( n = 17 blastomeres from 17 embryos, one-sample t test, **** P < 0.0001 significant deviation from 1). ( G ) Model for local regulation of ring constriction by the apical domain. In intact 16-cell embryos, apical polarity restricts the access of Anillin and p-Myosin to the contractile ring in circumferentially dividing outer cells, thereby slowing furrow ingression specifically on the side of the contractile ring that overlaps with the apical domain. Time is shown in minutes, where 0′ is anaphase onset. (Scale bars, 10 µm.) In the box plots, the center line represents the median, the bounds of the box represent upper and lower quartiles, the whiskers represent minimum and maximum values, and dots represent independent measurements.

Article Snippet: Primary antibodies used were PKC ζ anti-mouse 1:100 (sc-17781; Santa Cruz), Anillin anti-rabbit 1:300 (gift from Alisa Piekny, Concordia University, Montréal, Canada); p-Myosin light chain II anti-rabbit 1:100 (3671T; Cell Signaling Technology), and TEAD4 anti-mouse 1:100 (ab58310; Abcam).

Techniques: Immunofluorescence, Injection, Control, shRNA, Fluorescence, Two Tailed Test, Expressing, Cell Culture

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