cep55  (Santa Cruz Biotechnology)

Journal: The Journal of Cell Biology

Article Title: An ATM–Chk2–INCENP pathway activates the abscission checkpoint

doi: 10.1083/jcb.202008029

Figure Lengend Snippet: Mutation of Ser91 to alanine does not impair INCENP-association with CPC proteins. (A) Frequency of midbody stage cells. BE cells were released from a prometaphase block and treated with 10 µM Chk2 inhibitor II (inhII) in cytokinesis, or left untreated. Values represent mean ± SD from three independent experiments ( n > 900). (B) Accumulation of dead BE cells with time as determined by trypan blue staining. Mean ± SD from three independent experiments ( n = 6). (A and B) ***, P < 0.001 (ANOVA and Student’s t test). (C, D, G, and H) Localization of Mklp2 or Cep55 and mean intensity at the midbody or midbody center in BE cells. (E) GFP-Trap assay. BE cells were untransfected (untransf.) or transfected with GFP:INCENP. Precipitated (top) or total proteins (bottom) were analyzed by Western blotting. (F and L) Western blot analysis of total Cep55, ATM, actin and tubulin. (I) Total GFP and Mklp2 from . (J) Cartoon indicating the regions of human Mklp2 protein interacting with INCENP, Cep55 and myosin-II. Numbers show amino-acid residues. motor, kinesin motor domain. (K) Western blot analysis of total Mklp2 and actin in BE cells expressing GFP:Mklp2(ΔC90). (M) Specificity of the anti-phospho-ATM-Ser1981 (pATM-Ser1981) antibody. Tubulin values indicate midbody thickness. Insets show 1.6× magnification of the midbodies. (N and O) Phase-contrast live-cell microscopy analysis of HeLa tubulin:GFP cells treated with 10 µM KU-55933 in cytokinesis. Intercellular canals are shown by solid arrows. Time is from formation of an intercellular canal to canal cleavage (dotted arrows). Related to . Values represent mean ± SD from n cells. ***, P < 0.001 (Student’s t test). Scale bars, 5 µm. (P and Q) Frequency of prometaphase or bi/multinucleate BE cells. Values represent mean ± SD from three independent experiments ( n > 900). ns, not statistically significant. S, serine.

Article Snippet: Human Mre11 (a pool of three different siRNAs: 5′-CAG AAC AGA UGG CUA AUG A-3′, 5′-GUG AGG GAA UGG UCA CUA A-3′, and 5′-GUA GGG AAU UCU UCU GUU A-3′), ATM (a pool of three different siRNAs: 5′-GUA GCA ACA UAC UAC UCA A-3′, 5′-GCA ACA UAC UAC UCA AAG A-3′, and 5′-GCA ACC CAA UUA AUA UCA A-3′), Rad50 (a pool of three different siRNAs: 5′-CAA GGU UGC UCA AGA AAC A-3′, 5′-CCA ACC AAC UGA UGA AUG A-3′, and 5′-CAA CAG AAC UUG UGA ACA A-3′), and Cep55 (a pool of three different siRNAs: 5′-CUA CCG CAU UGC UUG AAC A-3′, 5′-CAG GUC CAG UUU CUU UAC A-3′, and 5′-GCA UGC UAG UGA AUC AUG U-3′) siRNAs were from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Blocking Assay, Staining, TRAP Assay, Transfection, Western Blot, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: An ATM–Chk2–INCENP pathway activates the abscission checkpoint

doi: 10.1083/jcb.202008029

Figure Lengend Snippet: GFP:INCENP binds to GST-Cep55 through Mklp2. (A) GFP-Trap kinase assay from BE cell extracts using histone H3 as substrate. GFP-associated histone H3-Ser10 phosphorylation (pH3-Ser10), immunoprecipitated Aurora B, and GFP were analyzed by Western blotting. Histone H3 was analyzed by Ponceau staining. 300 nM VX-680 was added to the kinase reaction to inhibit Aurora B catalytic activity. (B) Cep55:GFP colocalizes with INCENP at the midbody center in BE cells. (C–F) Localization and mean intensity of INCENP at the midbody or midbody center in BE cells. Mean ± SD from n cells. Values in control were set to 1. ***, P < 0.001 (Student’s t test). Tubulin values indicate midbody thickness. Insets show 1.6× magnification of the midbodies. Scale bars, 5 µm. (G) Immunoprecipitation from cytokinesis-enriched BE cell extracts. (H) Predicted experimental outcome. In the Western blot, the width of the lines indicates band signals intensity. (I and J) Cell lysates from BE cells were incubated with GST-Cep55 or GST. Associated proteins were detected by Western blotting. (K and N) Radiolabeled FL or truncated Mklp2 (ΔC90) was incubated with GST-Cep55 or GST. Phosphorimager ( 35 S, top) and Western blot analysis (WB; bottom) of the same gel. Asterisks, predicted molecular weights. (L and M) GFP-Trap assay. Precipitated (L) or input proteins (M) were analyzed by Western blotting. S, serine.

Article Snippet: Human Mre11 (a pool of three different siRNAs: 5′-CAG AAC AGA UGG CUA AUG A-3′, 5′-GUG AGG GAA UGG UCA CUA A-3′, and 5′-GUA GGG AAU UCU UCU GUU A-3′), ATM (a pool of three different siRNAs: 5′-GUA GCA ACA UAC UAC UCA A-3′, 5′-GCA ACA UAC UAC UCA AAG A-3′, and 5′-GCA ACC CAA UUA AUA UCA A-3′), Rad50 (a pool of three different siRNAs: 5′-CAA GGU UGC UCA AGA AAC A-3′, 5′-CCA ACC AAC UGA UGA AUG A-3′, and 5′-CAA CAG AAC UUG UGA ACA A-3′), and Cep55 (a pool of three different siRNAs: 5′-CUA CCG CAU UGC UUG AAC A-3′, 5′-CAG GUC CAG UUU CUU UAC A-3′, and 5′-GCA UGC UAG UGA AUC AUG U-3′) siRNAs were from Santa Cruz Biotechnology.

Techniques: Kinase Assay, Phospho-proteomics, Immunoprecipitation, Western Blot, Staining, Activity Assay, Control, Incubation, TRAP Assay