Journal: Developmental cell

Article Title: Brg1 governs a positive feedback circuit in the hair follicle for tissue regeneration and repair.

doi: 10.1016/j.devcel.2013.03.015

Figure Lengend Snippet: Figure 7. Partial Rescue of Cell Defects by Shh in BRG1-Deficient Human Bulge Cells (A–D) Growth of human bulge cells 48 hr after doxycycline (Dox) treatment and siCtrl (A and C) or siBRG1 (B and D) infection in the absence (A and B) or presence (C and D) of human recombinant SHH (rSHH). (E–H) BrdU incorporation of human bulge cells 48 hr after Dox treatment and siCtrl (E and G) or siBRG1 (F and H) infection in the absence (E and F) or presence (G and H) of rSHH. Arrows indicate BrdU-positive cells. (I) Quantification of BrdU incorporation in siCtrl ± rSHH and siBRG1 ± rSHH groups. (J and K) Quantitation of BRG1 and p27Kip1

Article Snippet: Rescue Experiments with Recombinant Human SHH Proteins and Lentivirus-Expressing SHH The siCtrl and siBrg1 bulge cells were treated with/without doxycycline and with/without 1 mg/ml active form of recombinant human SHH protein (R&D Systems) for 48 hr before analysis.

Techniques: Infection, Recombinant, BrdU Incorporation Assay, Quantitation Assay

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Pericentromeric cohesin is deprotected long before sister chromatids separate. During phosphorylation- and Wapl-dependent release of cohesin from chromosome arms, pericentromeric cohesin is protected by associated Sgo1-PP2A. Following the early mitotic degradation of Nek2a and cyclin A2, Sgo1-PP2A relocates to kinetochores. Yet, pericentromeric cohesion persists until securin and cyclin B1 are degraded and separase is activated. A requirement for Nek2a and cyclin A2 for prophase pathway signaling would explain why, in metaphase, Wapl can no longer release cohesin. ( B ) Nek2a and Cdk1/2-cyclin A2 are necessary for Wapl-dependent in vitro displacement of cohesin from chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, kinases, and inhibitors (BI2536, ZM-447439, and RO-3306), as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. ( C ) Wapl and the three kinases Nek2a, Cdk1/2-cyclin A2, and Aurora B-INCENP are sufficient to support the displacement of cohesin from isolated chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, Nek2a, Cdk1/2-cyclin A2, Cdk1-cyclin B1, aurora B, and/or Plk1, as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Purification, Sedimentation, Incubation, Recombinant, Western Blot, Isolation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Recombinant kinases are active as judged by the phosphorylation of model substrates. Plk1, aurora B-INCENP, Nek2a, Cdk1/2-cyclin A2, or Cdk1-cyclin B1 supplemented with their specific inhibitor or carrier solvent DMSO (−) were incubated with the corresponding model substrate in the presence of [γ 33 P]-ATP. Reactions were subjected to SDS-PAGE followed by autoradiography. BI BI2536, ZM ZM-447439, ST staurosporine, RO RO-3306, MBP myelin basic protein. ( B ) Recombinant Nek2a, Cdk1/2-cyclin A2, and Wapl are free of cyclin B1 and Plk1. The preparations of Nek2a, Cdk1/2-cyclin A2, Wapl, Cdk1-cyclin B1, and Plk1 used for the release assays were characterized by immunoblotting using the indicated antibodies. In vitro expressed (IVTT) cyclin B1 served as an additional control.

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Solvent, Incubation, SDS Page, Autoradiography, Western Blot, In Vitro, Control

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Experimental outline. DNA-mediated chromatin pull-down from G2-arrested HeLaK cells is followed by cohesin eviction experiments. ( B ) Cohesin is eluted from immobilized chromatin upon DNA-cleavage but not high salt treatment. Chromatin beads were treated with benzonase and/or high salt, and retained proteins were detected by immunoblotting. High-salt-washed chromatin (lane 6) was used for subsequent cohesin eviction experiments. ( C ) Wapl-dependent cohesin release in vitro requires Nek2a, Cdk1/2-cyclin A2, and Aurora B but not Cdk1-cyclin B1 and Plk1. Immobilized chromatin was combined with the indicated proteins. Following centrifugation, DNA-beads and supernatant were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Western Blot, In Vitro, Centrifugation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Nek2a and Cdk1/2-cyclin A2 target juxtaposed but distinct residues within the disordered, C-terminal domain of Pds5b. In vitro expressed Pds5b variants were incubated with the indicated kinases and inhibitors in the presence of [γ- 33 P]-ATP, subjected to SDS-PAGE, and analyzed by autoradiography and immunoblotting. Note that Pds5b is also phosphorylated by aurora B and, weakly, by Plk1. KD kinase-dead Nek2a-Lys37Met; IVTT coupled in vitro transcription-translation. ( B ) Graphical summary of Nek2a- and Cdk1/2-cyclin A2-targeted Pds5b residues and corresponding phosphorylation-resistant variants. ( C ) Ser1209 of Pds5b is phosphorylated by Nek2a but not Cdk1/2-cyclin A2. Following incubation of in vitro expressed Pds5b-WT or −3A with Nek2a or Cdk1/2-cyclin A2, samples were analyzed by immunoblotting using the indicated antibodies. Nek2a-Lys37Met (KD, kinase-dead) served as a negative control. ( D ) The spatiotemporal dynamics of Ser1209-phosphorylation correlate with the recruitment of Wapl to chromatin followed by cohesin release. HeLaK cells synchronously cycling through mitosis were subjected to time-resolved fractionation and immunoblotting. At the time of release from a RO-3306-mediated arrest, cultures were supplemented with a Nek2a inhibitor (NCL00017509) or carrier solvent (mock). .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Western Blot, Negative Control, Fractionation, Solvent

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Transduction, Sequencing, Protease Inhibitor, Membrane, Software