Journal: The EMBO Journal

Article Title: A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

doi: 10.1038/s44318-024-00104-6

Figure Lengend Snippet: ( A ) HeLa cells were transfected with control siRNA or CENP-U siRNA. At 48 h post-transfection, cells were treated with the solvent DMSO or MG132 for 8 h, then cell lysates were immunoblotted with antibodies for Wapl, SMC3, Scc1, and α-Tubulin. ( B – D ) HeLa cells were transfected with control siRNA, CENP-U siRNA, and/or Wapl siRNA. At 48 h post-transfection, cells were treated with MG132 for 8 h, then mitotic chromosome spreads were prepared and stained with the CENP-C antibody and DAPI. The percentage of cells in which the majority of sister chromatids was separated or unseparated was determined in 300 cells for each condition from three independent experiments, with statistics being analyzed for cells with separated chromatids ( B ). Example images are shown ( C ). Cell lysates were immunoblotted with antibodies for Wapl and α-Tubulin ( D ). ( E – H ) HeLa cells were transfected with the indicated siRNAs. At 48 h post-transfection, cells were treated with nocodazole for 3 h. Then mitotic chromosome spreads were prepared and stained with the CENP-C antibody and DAPI. The inter-KT distance was measured on over 1000 chromosomes in 20 cells ( E ). The means and individual data points from two independent experiments are plotted ( F ). Example images are shown ( G ). Cell lysates were immunoblotted with antibodies for Wapl and α-Tubulin ( H ). Data information: Statistics were performed using unpaired Student’s t -test ( B ) and one-way ANOVA ( E ). Means and SDs are shown ( B , E ). Scale bars, 10 µm ( C , G ). NS no significance ( B , E ). .

Article Snippet: Rabbit polyclonal antibodies used were GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), Scc1 (ab992, Abcam), SA2 (Rabbit mAb #5882, CST), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5B (A300-537A, A300-538A, Bethyl Laboratories), GST (G7781, Sigma), Wapl (A300-268A, Bethyl), Sororin (ab192237, Abcam), Flag (GenScript), H2ApT120 (Active motif).

Techniques: Transfection, Control, Solvent, Staining

Journal: The EMBO Journal

Article Title: A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

doi: 10.1038/s44318-024-00104-6

Figure Lengend Snippet: ( A ) Lysates prepared from asynchronous HeLa cells were subjected to pull-down with GST, GST-Scc1 (281–420), or GST-Scc1 (281–420)-SA2 (80–1060), followed by immunoblotting with antibodies for Wapl and GAPDH, and CBB staining. ( B ) Lysates prepared from HEK-293T cells transiently expressing Wapl-Flag in the forms of WT and the 3xEGE mutant were subjected to pull-down by GST or GST-Scc1 (281–420)-SA2 (80–1060), followed by immunoblotting with the antibody for the Flag-tag, and CBB staining. Irrelevant lanes were removed. ( C ) MBP-Wapl (1–630) in the forms of WT and the 3xEGE mutant were subjected to pull-down by GST, GST-Scc1 (281–420), or GST-Scc1 (281–420) -SA2 (80–1060), followed by immunoblotting with the antibody for MBP, and CBB staining. ( D , E ) U2OS-LacO cells transiently expressing the indicated proteins were stained with antibodies for the Flag-tag and Myc-tag, and DAPI. Example images are shown ( D ). The fluorescence intensity ratio of Wapl-Flag/EGFP at the LacO repeats was quantified in 30 cells for each condition ( E ). ( F , G ) U2OS-LacO cells transiently expressing the indicated proteins were stained with antibodies for the Flag-tag, Myc-tag, and DAPI. Example images are shown ( F ). The fluorescence intensity ratio of SFB-CENP-U/EGFP at the LacO repeats was quantified in 30 cells for each condition ( G ). Data information: The white arrows point to the LacO repeats ( D , F ). Scale bars, 10 µm ( D , F ). Statistics were performed using unpaired Student’s t -test ( E , G ). Means and SDs are shown ( E , G ). .

Article Snippet: Rabbit polyclonal antibodies used were GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), Scc1 (ab992, Abcam), SA2 (Rabbit mAb #5882, CST), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5B (A300-537A, A300-538A, Bethyl Laboratories), GST (G7781, Sigma), Wapl (A300-268A, Bethyl), Sororin (ab192237, Abcam), Flag (GenScript), H2ApT120 (Active motif).

Techniques: Western Blot, Staining, Expressing, Mutagenesis, FLAG-tag, Fluorescence

Journal: The EMBO Journal

Article Title: A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

doi: 10.1038/s44318-024-00104-6

Figure Lengend Snippet: ( A ) U2OS-LacO cells transiently expressing the indicated proteins and the mutants of EGFP-LacI-Scc1 (I337A/L341A) and Myc-SA2 (W334A) were stained with the antibody for the Myc-tag, and DAPI. Example images are shown. ( B ) U2OS-LacO cells transiently expressing the indicated proteins and the mutants of Myc-SA2 (F367A) and Myc-SA2 (F371A) were stained with the antibody for the Myc-tag and DAPI. Example images are shown. ( C ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and/or Myc-SA2 in the forms of WT and the indicated mutants were subjected to pull-down with GST or GST-CENP-U (1–60), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. ( D ) Lysates prepared from HEK-293T cells transiently co-expressing Scc1-GFP and Myc-SA2 (WT and the indicated mutants) were subjected to pull-down with GST or GST-CENP-U (1-60), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. Irrelevant lanes were removed. ( E ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and/or Myc-SA2 in the forms of WT and the indicated mutants were subjected to pull-down with MBP or MBP-Wapl (1–630), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. ( F ) Lysates prepared from HEK-293T cells transiently co-expressing Scc1-GFP and Myc-SA2 (WT and the indicated mutants) were subjected to pull-down with MBP or MBP-Wapl (1–630), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. ( G ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with GST-CENP-U (1–60) in the presence of increased amount of eluted MBP-Wapl (1–630) protein, followed by immunoblotting with antibodies for MBP, GFP and the Myc-tag, and CBB staining. ( H ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with GST-CENP-U (1–60) in the presence of increased amounts of eluted MBP-Wapl (1–630) protein (WT or the 3xEGE mutant), followed by immunoblotting with antibodies for MBP and the Myc-tag, and CBB staining. ( I ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with MBP-Wapl (1–630) in the presence of increased amounts of eluted GST-CENP-U (1–60) protein (WT or the ADA mutant), followed by immunoblotting with antibodies for GST, GFP, and the Myc-tag, and CBB staining. Data information: The white arrows point to the LacO repeats ( A , B ). Scale bars, 10 µm ( A , B ). .

Article Snippet: Rabbit polyclonal antibodies used were GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), Scc1 (ab992, Abcam), SA2 (Rabbit mAb #5882, CST), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5B (A300-537A, A300-538A, Bethyl Laboratories), GST (G7781, Sigma), Wapl (A300-268A, Bethyl), Sororin (ab192237, Abcam), Flag (GenScript), H2ApT120 (Active motif).

Techniques: Expressing, Staining, Western Blot, Mutagenesis

Journal: PLoS ONE

Article Title: Short-term succinic acid treatment mitigates cerebellar mitochondrial OXPHOS dysfunction, neurodegeneration and ataxia in a Purkinje-specific spinocerebellar ataxia type 1 (SCA1) mouse model

doi: 10.1371/journal.pone.0188425

Figure Lengend Snippet: (A) Representative calbindin-stained cerebellar primary fissure images from untreated wild type, untreated B05 and treated B05 mice. (B) Molecular layer thickness, a measure of calbindin-positive Purkinje dendritic length, in treated and untreated wild type and B05 cerebellum. (C) Representative ATXN1-stained cerebellar primary fissure images from untreated control transgenic (A02), untreated B05 and treated B05 mice. (D) Purkinje nuclei counts in treated and untreated A02 and B05 cerebellum. Error bars represent means ± SEM. Two-way ANOVA,* P < 0.05, ** P < 0.01.

Article Snippet: Following an established protocol [ ], epitopes were unmasked by boiling three times for 15 seconds each in 0.01 M urea, blocked for 1 hour in 2% normal donkey serum and 0.3% Triton X-100 in 1X PBS, and incubated for 48 hours at 4°C in blocking solution containing goat calbindin antibody (SC-7691, Santa Cruz) at 1:500.

Techniques: Staining, Control, Transgenic Assay

Journal: Autophagy

Article Title: Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability

doi: 10.1080/15548627.2020.1764727

Figure Lengend Snippet: List of primary and secondary antibodies used in this study

Article Snippet: WAPL , WB 1/1000 IF 1/50 , Bethyl Lab. Santa Cruz Biotechnology , A300-268A-M sc-365189.

Techniques: