lambda protein phosphatase kit  (New England Biolabs)

Journal: Acta Neuropathologica Communications

Article Title: Characterisation of tau in the human and rodent enteric nervous system under physiological conditions and in tauopathy

doi: 10.1186/s40478-018-0568-3

Figure Lengend Snippet: Poor susceptibility of tau to dephosphorylation in mature ENS. a Sigmoid colon biopsies lysates from 2 control subjects (#183, 3 first lanes and #208, 2 last lanes) were subjected to immunoblot analysis using TAU-5, AT8, PHF-1 and Tau-1 and antibodies. Lysates were treated with (+ for 1 h and ++ for 3 h) or without (−) lambda phosphatase before immunoblotting. The effectiveness of dephosphorylation was confirmed by phospho-ERK immunoblot (P-ERK immunoblot). b Antibodies against total tau (Tau antibody A0024) and tau phosphorylated at serine 202 (CP13) (both shown in red) were used to detect tau in the myenteric plexus of the proximal colon from htau mice. eGFP expression is shown in green, together with merged images including Hoechst 33,342 nuclear labelling (blue). Scale bars are 100 μm. N = 3. c . Brain and proximal colon homogenates from htau mice were treated with or without lambda phosphatase (+ or -) and immunoblotted with pan-Tau antibody A0024. A recombinant human tau ladder was included on each blot. White lines indicate rearrangement of lanes within the same blot for clarity. Data and images are representative of three independent experiments

Article Snippet: Samples were diluted to 1.0 mg/mL protein using homogenisation buffer and incubated with 20 U/μL lambda phosphatase in MnCl2 and enzyme buffer as supplied with the lambda protein phosphatase kit (New England Biolabs, Ipswich, MA, USA) for 3 h at 30 °C.

Techniques: De-Phosphorylation Assay, Mouse Assay, Expressing, Recombinant

Journal: Acta Neuropathologica Communications

Article Title: Characterisation of tau in the human and rodent enteric nervous system under physiological conditions and in tauopathy

doi: 10.1186/s40478-018-0568-3

Figure Lengend Snippet: Tau isoform profile and phosphorylation state in rat primary culture of ENS. a Lysates of rat primary ENS and CNS cultures were subjected to immunoblot analysis using the pan-Tau antibody A0024 and the isoform specific antibodies 3R and 4R. b Primary culture lysates were treated with (+) or without (−) lambda phosphatase before immunoblot analysis with the pan-Tau antibody A0024 and the isoform specific antibody against 3R-tau. PGP9.5 was used as a loading control. c Primary culture of rat ENS were treated (+) or not (−) with a cocktail of 3 phosphatase inhibitors including 1 μM okadaic acid, 1 μM ciclosporine A and 6.75 μM sanguinarine (Ppase inhibitors) for 1 h. Fifteen μg of cell lysates were subjected to immunoblot analysis using Tau-1, AT8 and PHF-1 antibodies. IB is for immunoblot. The results shown in ( a ), ( b ) and ( c ) are representative of 2, 4 and 3 independent experiments, respectively

Article Snippet: Samples were diluted to 1.0 mg/mL protein using homogenisation buffer and incubated with 20 U/μL lambda phosphatase in MnCl2 and enzyme buffer as supplied with the lambda protein phosphatase kit (New England Biolabs, Ipswich, MA, USA) for 3 h at 30 °C.

Techniques:

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: BIN1 phosphorylation at T348 regulates BIN1–Tau interaction by modulating open/closed conformation of BIN1. a Alignment of Amphiphysin 1 and BIN1iso1; domains not to scale. The underlined sequence indicates the BIN1 PRD sequence interacting with the BIN1 SH3 domain. b Lambda protein phosphatase (λ-PP) treatment dephosphorylates BIN1; 2 lanes per condition. c. In vitro phosphorylation assays with recombinant proteins show that Cdk2 and Cdk5 phosphorylate BIN1 at T348. Also see Fig. S4. d , e Immunoblots and quantification showing the effects of U0126 and CsA (10 μM; 2.5 h) on BIN1 and Tau phosphorylation. Inset shows the effect of 10 nM CsA on BIN1 phosphorylation. Mean ± SD from 3 independent experiments. One-way ANOVA and paired t test; * p

Article Snippet: Lambda protein phosphatase assay Crude protein extracts were incubated with Lambda protein phosphatase (New England Biolabs; Ipswich, MA), following supplier’s instructions with minor changes.

Techniques: Sequencing, In Vitro, Recombinant, Western Blot

Journal: bioRxiv

Article Title: DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection

doi: 10.1101/2021.02.08.430376

Figure Lengend Snippet: H2AS15ph is locally induced upon DNA double strand break formation and spread over several kilobases. (A) H2A-S15ph signal increased when yeast cells are treated with DNA damaging agent, MMS. Western blot analysis was performed with 300ng of native chromatin purified from yeast cells treated with DMSO (untreated control) and MMS. Anti-H2AS129ph was used as positive control for the MMS treatment. Antibodies against histone H2B and H4 were used as loading control. (B) Anti-H2AS15ph is specific for recognizing a phosphorylation mark on H2A. Western blot analysis was performed with 300ng of native chromatin purified form MMS treated yeast cells after incubation with or without lambda protein phosphatase (λPP). Lane with λPP treatment showed a significant decrease in signal for H2A-S15ph. Histone H4 was used as loading control. (C) H2AS15ph spreads on both sides of the HO endonuclease induced single DSB at MAT locus. Signal for H2AS15ph showed enrichment after the induction of the DSB in wild type but not in H2A-S15A . ChIP-qPCR was performed as described in Figure 1D . Fold enrichment was calculated as ratio of %IP/ input normalized on total H4 for histone occupancy at indicated loci around DSB at MAT locus to the signal at negative-control locus intergenicV. (D, E) Kinetics of H2S15ph and H2AS129ph accumulation after the induction of the HO-DSB. ChIP samples were analyzed after times 0, 30min, 1hr and 3hrs. Anti-H4 signal was used to normalize for histone occupancy. ChIP-qPCR was performed as described in Figure 1D .

Article Snippet: Lambda protein phosphatase (λPP) (NEB) kit was used.

Techniques: Western Blot, Purification, Positive Control, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

Journal: Journal of Cell Science

Article Title: Phosphorylation of the RNA-binding protein Zfs1 modulates sexual differentiation in fission yeast

doi: 10.1242/jcs.208066

Figure Lengend Snippet: Nitrogen depletion and inhibition of TOR signaling promote Zfs1 phosphorylation . (A) Western blot of Zfs1 protein during incubation in medium without nitrogen. Cells were grown in minimal EMM to mid-log phase, then washed with medium without nitrogen and incubated in this for the indicated times. (B) Protein extracts from cells treated as in A were incubated with λ-protein phosphatase (see Materials and Methods). (C) Torin-1 inhibition of TOR complexes stimulates Zfs1 hyper-phosphorylation. Cells growing in EMM were incubated with 10 µM Torin-1 or the same volume of DMSO for the indicated times. (D) The AKT1-related Gad8 kinase is required for hyper-phosphorylation of Zfs1. Cells were grown in EMM and then washed and incubated in EMM without nitrogen for 2 h.

Article Snippet: For protein dephosphorylation assays, up to 150 µg of protein extracted with beating buffer without EDTA and phosphatase inhibitors, was treated with 400 U of λ-protein phosphatase (New England Biolabs) for 50 min at 30°C.

Techniques: Inhibition, Western Blot, Incubation