Journal: PLOS One

Article Title: Identification of CAND1 as a DNA-dependent protein kinase-regulated coactivator of androgen receptor and the ARv7 splice variant

doi: 10.1371/journal.pone.0349130

Figure Lengend Snippet: (A) After standard ARE pulldowns, the AR-DNA-bound complexes were washed and resuspended in 1x kinase buffer with/without ATP at 30 o C for listed times. Gel mobility shifts in AR were detected as a function of ATP addition. All pulldowns had 100 nM R1881 present. Proteins were analyzed by immunoblotting. Lanes 3 and 4 represent the standard AR-ARE pulldown. (B) To test if the above AR gel mobility shifts were reflective of phosphorylation events, the AR-CoR complexes were treated with ATP or AMP-PNP (non-hydrolyzable analogue) for 30 min at 30 o C. The DNA-PK inhibitor NU7441 or λ protein phosphatase (LPP) were pre-incubated for 5 min before adding ATP. LPP buffer was used for the LPP control reaction. (C) DNA-PK plays a role in phosphorylating AR. ATP treatment of the AR-CoR complexes using HeLa NE where DNA-PKcs was immunodepleted reveals a reduction of AR gel mobility shifts ( left panel) with the loss of DNA-PK. Proteins were assayed by immunoblotting and the level of unphosphorylated AR was quantified by Image J after normalization to the loading control HDAC1 ( right panel). P-value here = 0.00011. (D) DNA-bound AR or ARv7 was incubated with purified heterotrimeric DNA-PK (i.e., DNA-PKcs and Ku70/80) and then treated (-/+) ATP (-/+) NU7441 to demonstrate direct phosphorylation of AR/ARv7 by DNA-PK. Gel mobility shifts were assayed by immunoblotting. (E) ATP treatment (-/+) NU7441 pre-treatment demonstrates that DNA-PK enzymatic activity is needed for stabilizing CAND1 and ANP32A in the AR/ARv7 complexes. Auto-phosphorylation of DNA-PKcs at serine 2056 (pS2056) is a marker of DNA-PK activity and its inhibition by NU7441. Proteins were assayed by immunoblotting. ARv7 was detected here with the N-20 antibody. (F) HeLa NE used in 3xARE-E4 cell-free transcription assays was pre-treated (-/+) NU7441 to determine effects on AR and ARv7 transcriptional activity. E4 mRNA transcript levels were measured by RT-qPCR. In panels A-C and E, HDAC1 was used as a loading control and ARE pulldowns were conducted using HeLa NE as listed. All AR reactions had 100 nM R1881 present. Short exp., Medium exp., and Long exp. denote short, medium, and long exposures during x-ray film development. P-values: for ARv7, p = 0.00015; for AR, p = 0.000035. **** p < 0.0005, ***** p < 0.0001.

Article Snippet: DNA-PK inhibitor NU7441 was purchased from Tocris Bioscience. λ protein phosphatase (LPP) was purchased from New England Biolabs.

Techniques: Western Blot, Phospho-proteomics, Incubation, Control, Purification, Activity Assay, Marker, Inhibition, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Ultrasonic Reporter of Kinase Activity

doi: 10.64898/2025.11.30.691048

Figure Lengend Snippet: a) xAM images of UReKA1 and UReKA2 incubated with PKA at various timepoints at 634kPa (for maximal signal). b) Normalized nonlinear US signal of UReKA1 (blue) and UReKA2 (orange) over time (n=3). c) Normalized phosphorylated fraction of UReKA1 (blue) and UReKA2 (orange) over time (n=1). d) Nonlinear ultrasound images (left) and signal (right) of UReKA1 incubated with PKA and phosphorylated UReKA1 incubated with LPP with or without buoyancy purification. (n=2). e-f) Schematics of the proposed molecular mechanism of UReKA with reversible phosphorylation reaction for the phosphorylation as forward reaction (e) and dephosphorylation of GvpC as reverse reaction (f).

Article Snippet: For dephosphorylation assays, 10 units of LPP (P0753L; NEB) per μL of engineered GVs at OD500 = 10 were mixed with PMP Buffer (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, pH 7.5) and 1 mM MnCl 2 , followed by incubation with slow rotation at 37°C for 10–12 hours.

Techniques: Incubation, Purification, Phospho-proteomics, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: Rare bioactive tau oligomers from Alzheimer brain support both templated misfolding and fibril formation

doi: 10.1101/2025.09.24.678227

Figure Lengend Snippet: A) Schematic representation of HMW tau dephosphorylation by the Lambda Protein Phosphatase (LPP). B) Seeding activity assay using FRET reporter HEK cell; the integrated FRET intensity was quantified by flow cytometry. Data represents mean ± S.D. of three individual experiments performed in triplicate with HMW Tau isolated from the AD case 1971. Results were analyzed using the unpaired student t-test, ** for p < 0.005. C), D) Dot blot quantification of HMW tau, originating from the same case as panel B, treated or not with Lambda PP and probed with the anti-phospho tau AT8 or the total tau antibody D5D8N.

Article Snippet: HMW tau was subjected to dephosphorylation using Lambda Protein Phosphatase (LPP) kit (New England Biolabs (NEB), #P0753S).

Techniques: De-Phosphorylation Assay, Activity Assay, Flow Cytometry, Isolation, Dot Blot

Journal: Acta Neuropathologica

Article Title: Synaptotoxic effects of extracellular tau are mediated by its microtubule-binding region

doi: 10.1007/s00401-025-02897-0

Figure Lengend Snippet: MTBR/R’ is required for acute plasticity disruption by synaptotoxic tau in AD brain extracts. a Aqueous extracts from the brains of two people with AD (AD1 and AD6) were analysed by Western blotting in the presence or absence of lambda protein phosphatase (± LPP) using the monoclonal anti-tau antibodies Gen2A and Gen2B. Migration of SDS-PAGE molecular weight standards (in kDa) is indicated on the left. The bracket on the right highlights the position of full-length tau whereas arrows indicate likely tau fragments detected by both mAbs. It should be noted that AD6 extract had much higher total tau concentration as measured by immunoassay . b Unlike isotype control IgG, anaesthetised rats co-injected with AD1 extract (10 µL) and either Gen2A or Gen2B antibodies (2.5 µg i.c.v.) maintained normal hippocampal LTP. c Similarly, robust LTP was induced when Gen2B mAb (2.5 µg i.c.v.) was co-administered with the extract AD6 (10 µL). Left-hand panels in b, c show the time course of LTP. Summary bar charts of LTP magnitude during the last 10 min are displayed in the right-hand panels. Values are mean ± SEM. ## p < 0.01, ### p < 0.001 compared with pre-HFS, paired t -test; *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by Bonferroni’s multiple-comparison tests in b and unpaired t -test in c

Article Snippet: Aqueous extracts from the brains of two people with AD (AD1 and AD6) were analysed for tau protein by western immunoblotting in the presence or absence of lambda protein phosphatase (LPP) concentrate supplied at 400,000 units/ml, diluted 1:50 to 8000 units/ml for final reaction as per kit recommendation (New England Biolabs P0753S).

Techniques: Disruption, Western Blot, Migration, SDS Page, Molecular Weight, Concentration Assay, Control, Injection, Comparison