Dataset Information

Autophosphorylation of Tousled-like kinases (TLK1 and TLK2) regulates recruitment to damaged chromatin via PCNA interaction (Tandem Affinity Purification coupled to LC-MS/MS of SFB-TLK1 WT and TLK2 WT)

ABSTRACT: S-protein-2xFLAG-Streptavidin binding peptide (SFB)-tagged TLK1 or SFB-TLK2 were transfected into 4x 15 cm plates of HEK293T cells. Cells were harvested 24 hours after transfection using NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% NP-40) supplemented with 2 µg/mL aprotinin (Thermo, AAJ60237MB) and 5 µg/mL pepstatin A (Thermo, PI78432) at 4ºC for 20 minutes. Lysates were centrifuged at 9,000 x g, 4ºC for 20 minutes to yield supernatant as soluble fraction. Soluble fractions were incubated with streptavidin sepharose (200 µl) (GE Healthcare, GE17-5113-01) at 4ºC for 1 hour followed by washing with NETN buffer three times. The protein complexes were eluted with 2 mg/mL biotin at 4ºC for 1 hour. The eluents were then incubated with S-protein agarose (EMD Millipore, 69704-3) overnight at 4ºC, washed three times with NETN buffer, and eluted in 1x Laemmli buffer.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kirk West

LAB HEAD: Justin W. Leung

PROVIDER: PXD047996 | Pride | 2025-01-02

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Leung_052319_TLK1_S.raw	Raw
	Leung_052319_TLK1_S.raw__F054684_.mzid.gz	Mzid
	Leung_052319_TLK1_S.raw__F054684_.mzid_Leung_052319_TLK1_S.raw__F054684_.MGF	Mzid
	Leung_052319_TLK2_S.raw	Raw
	Leung_052319_TLK2_S.raw__F054686_.mzid.gz	Mzid

Items per page:

1 - 5 of 7

Publications

Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction.

West Kirk L KL Nguyen Tram T N TTN Tengler Kyle A KA Kreiling Natasha N Raney Kevin D KD Ghosal Gargi G Leung Justin W JW

Nucleic acids research 20241227

Tousled-like kinases 1 and 2 (TLK1 and 2) are cell cycle-regulated serine/threonine kinases that are involved in multiple biological processes. Mutation of TLK1 and 2 confer neurodegenerative diseases. Recent studies demonstrate that TLK1 and 2 are involved in DNA repair. However, there is no direct evidence that TLK1 and 2 function at DNA damage sites. Here, we show that both TLK1 and TLK2 are hyper-autophosphorylated at their N-termini, at least in part, mediated by their homo- or hetero- dime ...[more]

PMID: 39727191

Similar Datasets

Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.