Journal: Journal of Cell Science

Article Title: An acentrosomal aster with atypical microtubule polarity recruits cytokinesis signals to its center in Xenopus egg extracts

doi: 10.1242/jcs.263766

Figure Lengend Snippet: Formation of the noncanonical aster requires the microtubule motor MKLP2 and Aurora kinase B activity. (A) Confocal images of microtubule organization in control extracts and extracts with 100 µM MKLP2 inhibitor paprotrain. n =4. Each image is a maximum-intensity projection of nine confocal planes spanning 24 μm of depth. (B) Confocal images of microtubule dynamics in control extracts (top row) and extracts with 40 µM Aurora kinase B inhibitor barasertib (bottom row). n =6. Each image is a maximum-intensity projection of nine confocal planes spanning 16 µm of depth. For both A and B, imaging started at an arbitrary time point when asters had just begun to form in the untreated extracts. (C) Widefield epifluorescence images of microtubule organization in control extracts and extracts with 100 µM kinesin Eg5 inhibitor STLC. n =10. (D) Confocal images of microtubule organization in control extracts and extracts with 2 µM dynein inhibitor GST–p150-CC1. n =6. (E) Widefield epifluorescence images of microtubule and ER organization in control extracts and extracts with 0.68 µM GST–p150-CC1. n =2.

Article Snippet: The chicken DCTN1 p150Glued AA 217–548 (CC1) was from the plasmid pVEX-CC1 (Addgene plasmid 74170; http://n2t.net/addgene:74170 ; RRID:Addgene_74170; deposited by Trina Schroer).

Techniques: Activity Assay, Control, Imaging

Journal: Journal of Cell Science

Article Title: An acentrosomal aster with atypical microtubule polarity recruits cytokinesis signals to its center in Xenopus egg extracts

doi: 10.1242/jcs.263766

Figure Lengend Snippet: Noncanonical asters can merge. (A) Confocal time-lapse montage of microtubule and EB1–GFP dynamics in egg extracts, showing that the centers of two noncanonical asters merged with each another spontaneously, and that the EB1–GFP-enriched regions at the centers also merged. Each image is a maximum-intensity projection of four confocal planes spanning 6 µm of depth. Imaging started at an arbitrary time point after the asters had formed but had not merged. The plot below each image is the fluorescence intensity profile along a 1.65 µm thick, 22.8 µm long line segment (yellow dashed rectangle) that starts at the bottom left and ends at the top right. For each point on the curve in the plot, the horizontal coordinate is the distance from the start of the line segment, and the vertical coordinate is the average fluorescence intensity of the pixels across the width of the line segment at that distance (a.u., arbitrary units). The black arrows indicate intensity peaks for microtubule (second row) and EB1–GFP (fourth row) fluorescence at the aster centers. n =9. (B) Confocal images of microtubules in control and GST–p150-CC1-treated extracts, showing that noncanonical asters still merged when dynein was inhibited by 2 µM GST–p150-CC1. n =2.

Article Snippet: The chicken DCTN1 p150Glued AA 217–548 (CC1) was from the plasmid pVEX-CC1 (Addgene plasmid 74170; http://n2t.net/addgene:74170 ; RRID:Addgene_74170; deposited by Trina Schroer).

Techniques: Imaging, Fluorescence, Control

Journal: The Journal of Neuroscience

Article Title: Axonal Dynactin p150 Glued Transports Caspase-8 to Drive Retrograde Olfactory Receptor Neuron Apoptosis

doi: 10.1523/JNEUROSCI.0707-05.2005

Figure Lengend Snippet: Interaction of caspase-8 and dynactin p150Glued is mediated by the dynactin CC1 domain. A, Dynactin p150Glued contains a pDED within its first coiled-coil domain (CC1), which could form the basis for an interaction with the DEDs of procaspase-8. The first and last amino acids of the pDED are indicated on the left and right of the sequence, whereas the region corresponding to the fifth helix (α5) of the pDED containing the critical lysine (K) residue that differentiates a pDED from a classical DED, which would have a hydrophobic residue at the equivalent position, is underlined. B, Transfected, mutated (Cys-Ala in catalytic domain) flag-tagged procaspase-8 (ProC8) (C-A) was immunoprecipitated and found to be complexed with endogenous dynactin p150Glued from extracts of HEK293T cells (lane 2). The flag-tagged procaspase-8 (C-A):dynactin p150Glued interaction was reduced (arrow) by co-overexpression of the dynactin CC1 domain but not the CC2 domain (lanes 3, 4; B, beads alone control). Western blots confirm expression of flagged caspase-8 in whole-cell extracts (WCE) of HEK293 cells. C, An anti-dynactin mAb immunoprecipitated cold (unlabeled) in vitro translated dynactin p150Glued with 35S-labeled in vitro translated procaspase-8 (proC8) (Mock, mock caspase-8 transfection; B, beads alone control). D, Flag-tagged procaspase-8 (ProC8) (C-A) will coimmunoprecipitate with the dynactin CC1 domain but not the CC2 domain in HEK293T cells. E, Binding of dynactin p150Glued (p150) or the CC1 domain to caspase-8 did not require the prodomain. Immunoprecipitation of flag-tagged caspase-8 lacking its prodomain (caspase-8 Δprodomain) coimmunoprecipitated both full-length dynactin p150Glued or CC1 but not CC2 in overexpressing HEK293T cell extracts.

Article Snippet: DNA constructs Expression constructs for dynactin p150 Glued , CC1 (amino acids 217-548), and CC2 (amino acids 926-1049) were kindly provided by Dr. T. Schroer (Johns Hopkins University) and were described by Quintyne et al. ( 1999 ).

Techniques: Sequencing, Residue, Transfection, Immunoprecipitation, Over Expression, Control, Western Blot, Expressing, In Vitro, Labeling, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 blockade attenuates white matter inflammation and oligodendrocyte loss after progressive systemic lipopolysaccharide exposure in near-term fetal sheep

doi: 10.1186/s12974-021-02238-4

Figure Lengend Snippet: White matter immunohistochemistry. Glial fibrillary acidic protein (GFAP+), ionised calcium binding adaptor molecule (Iba-1+), amoeboid (Iba-1+) microglia, IL-1β immunoreactivity score, caspase 3, oligodendrocyte transcriptase factor-2 (Olig-2+) and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase+) cell counts, % area fraction of CNPase staining, anti-adenomatous polyposis coli clone (CC1+) cell counts and % area fraction of myelin basic protein (MBP) in the periventricular white matter (PVWM), first and second intragyral white matter tracts (IGWM 1 and IGWM 2) in control (blue circles, n = 9), LPS+vehicle (red circles, n = 8) and LPS+IL-1Ra (green circles, n = 9) groups. Data are means ± SE. * P < 0.05 vs. control, # P = 0.06 vs. control

Article Snippet: Sections were labelled with 1:200 rabbit anti-glial fibrillary acidic protein (GFAP; Abcam, cat#: ab68428), 1:200 rabbit anti-ionised calcium binding adaptor molecule 1 (Iba-1, Abcam, cat#: ab153696), 1:200 rabbit anti-oligodendrocyte transcription factor 2 (Olig-2, for oligodendrocytes at all stages of development; Abcam, cat#: ab42453;), 1:200 mouse anti-cyclic nucleotide 3′ phosphodiesterase (CNPase, for immature and mature oligodendrocytes; Abcam, cat#: ab6319), 1:200 rat anti-myelin basic protein (MBP, for myelin density; MerkMillipore, cat#: MAB395), 1:200 mouse anti-adenomatous polyposis coli clone (CC1, for mature oligodendrocytes, MerckMillipore, cat#: OP80-100UG), 1:800 rabbit anti-cleaved caspase3 (Abcam, cat#: ab2302), 1:200 rabbit anti-neuronal nuclei (NeuN, Abcam, cat#: ab177487) and 1:250 rabbit anti-IL-1β (cat#: NB600-633, Novus, CO, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Binding Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 blockade attenuates white matter inflammation and oligodendrocyte loss after progressive systemic lipopolysaccharide exposure in near-term fetal sheep

doi: 10.1186/s12974-021-02238-4

Figure Lengend Snippet: Representative photomicrographs showing positive staining of GFAP, Iba-1, IL-1β, caspase 3, Olig-2, CC1, MBP and CNPase in the periventricular white matter tracts. Scale bar = 100 μm. Arrows in the Iba-1 photomicrographs indicate microglia displaying a resting ramified phenotype, characterised by a small cell body with > 1 branching process. Arrowheads indicate microglia displaying an amoeboid morphology, characterised by a large cell body with ≤ 1 branching process. Arrows in the caspase-3 photomicrographs indicate positive cells displaying both staining and apoptotic bodies

Article Snippet: Sections were labelled with 1:200 rabbit anti-glial fibrillary acidic protein (GFAP; Abcam, cat#: ab68428), 1:200 rabbit anti-ionised calcium binding adaptor molecule 1 (Iba-1, Abcam, cat#: ab153696), 1:200 rabbit anti-oligodendrocyte transcription factor 2 (Olig-2, for oligodendrocytes at all stages of development; Abcam, cat#: ab42453;), 1:200 mouse anti-cyclic nucleotide 3′ phosphodiesterase (CNPase, for immature and mature oligodendrocytes; Abcam, cat#: ab6319), 1:200 rat anti-myelin basic protein (MBP, for myelin density; MerkMillipore, cat#: MAB395), 1:200 mouse anti-adenomatous polyposis coli clone (CC1, for mature oligodendrocytes, MerckMillipore, cat#: OP80-100UG), 1:800 rabbit anti-cleaved caspase3 (Abcam, cat#: ab2302), 1:200 rabbit anti-neuronal nuclei (NeuN, Abcam, cat#: ab177487) and 1:250 rabbit anti-IL-1β (cat#: NB600-633, Novus, CO, USA) overnight at 4 °C.

Techniques: Staining

Journal: Journal of Translational Medicine

Article Title: Immune cell infiltration and inflammatory landscape in primary brain tumours

doi: 10.1186/s12967-024-05309-1

Figure Lengend Snippet: List of the antibodies. Technical specifications of the antibodies used for the IHC staining

Article Snippet: TG2 , Zedira, Darmstadt, Germany , Mouse monoclonal , XTG17 , Pre-diluted , a CC1.

Techniques:

Journal: Molecular Psychiatry

Article Title: Shank3 related oligodendrocyte alterations in autism are restored by Erk pathway inhibition

doi: 10.1038/s41380-025-03333-1

Figure Lengend Snippet: Coronal brain sections from Shank3 WT and KO mice of corpus callosum (CC) have been used for all analyses. A Brain sections from P7 WT and Shank3KO mice stained for MBP and MBP RNA scope. MBP protein and RNA intensity were normalized to mean value. MBP positive cells were shown in the percentage of DAPI. Mean ± SD. Student’s Unpaired t test, p** < 0.01, n = 3 animals. Scale bar = 200 μm. B Immunostaining for MBP and NFH SMI32 and analysis of myelinating and non-myelinating oligodendrocytes in P7 CC. Myelinating and non-myelinating cells were shown in the percentage of total MBP positive cells. Mean ± SD. Student’s Unpaired t test, p**** < 0.0001, n = 3 animals. Scale bar = 100 μm. C P7, P21 and P140 brain section were stained for Olig2 and CC1. Cell number of Olig2 + CC1+ cells were analysed in CC. Data were shown in the percentage of DAPI and Olig2 respectively. Mean ± SD. Student’s Unpaired t test, p* < 0.05, n = 3 animals. Scale bar = 20 μm. D P7 brain section were stained for Ki67, Iba1 and GFAP and proliferation of Ki67+cells was analysed in medial and lateral CC separately. Ki67 positive cells were shown in the percentage of DAPI. Mean ± SD. Student’s Unpaired t test, p* < 0.05, n = 3 animals. Scale bar = 20 μm.

Article Snippet: CC1 (ms) , Merck , Germany , MABC200 , IHC , 1:500 , 10% DS + 5% FBS + 0.3% Triton in PBS-/-.

Techniques: Staining, RNAscope, Immunostaining