lambda phosphatase treatments  (New England Biolabs)

Journal: bioRxiv

Article Title: Two orthogonal MAP3K-driven pathways of NLRP1 inflammasome activation revealed by poisonous beetles

doi: 10.64898/2026.01.23.701189

Figure Lengend Snippet: TAK1 directly phosphorylates the NLRP1 DR at specific sites and requires a specific TST motif for NLRP1 inflammasome activation. A. Rhodamine labelled recombinant SNAP-tagged NLRP1-DR following PhosTag-SDS-Agarose-PAGE. Recombinant NLRP1-DR was incubated with recombinant ZAKα, p38α, and TAK1-TAB1 fusion in a standard kinase reaction for 60 minutes and labelled with SNAP ligand fluorescence (TMR-STAR). Lambda protein phosphatase or a negative control was spiked into each reaction, as indicated. Phosphorylated vs unphosphorylated species are indicated. B. IL-1β ELISA of NLRP1 KO N/TERTs overexpressing GFP_NLRP1-86-C_FLAG (WT), SATA 1-4, or STless mutants following treatment with 50 μM cantharidin or 1 μM anisomycin. Cell culture media was harvested after 8 hours of treatment. C. Recombinant SNAP-tagged NLRP1-DR was incubated with recombinant TAK1-TAB1 fusion or ZAKα in a standard kinase reaction for 60 minutes and mass spectrometry was used to detect phosphorylated peptides. Phosphorylated residues detected by mass spectrometry are indicated with solid dots while residues identified from the following add-back analysis are indicated with clear dots. Residues phosphorylated by TAK1 and ZAKα are indicated with blue and red respectively. D. Rhodamine labelled SNAP-tagged NLRP1-DR ‘Add-back’ mutants following PhosTag-SDS-Agarose-PAGE. In the STless background, indicated alanine residues were mutated back to serine or threonine. These recombinantly expressed mutants were incubated with recombinant TAK1-TAB1 fusion or ZAKα in a standard in vitro kinase reaction for 60 minutes. Phosphorylated vs unphosphorylated species for each protein is indicated. The crucial “TZ2” motif is highlighted in red. E. Immunoblot of cleaved PARP1, GSDMD (full length and cleaved), GSDME (full length and cleaved), GAPDH, Pro-IL-1β, cleaved IL-1β, and IL-18 (full length and cleaved) or F. IL-18 and IL-1β ELISA from NLRP1 KO N/TERTs overexpressing GFP_NLRP1-86-C_FLAG (WT), TZ2 mutant, or GFP control following treatment with 1 μM anisomycin, DMSO (vehicle control), and 50 μM cantharidin. Cells, floaters and cell culture media were harvested after 8 hours of treatment. Error bars represent SEM from three technical replicates, where one replicate refers to an independent sample. Significance values were calculated based on one-way ANOVA followed by Dunnett’s test for multiple pairwise comparisons (B) or two-way ANOVA followed by Sidak’s test for multiple pairwise comparisons (F). ns, nonsignificant; *P < 0.05; ****P < 0.0001.

Article Snippet: For lambda phosphatase treatments, a final concentration of 1X NEBuffer for Protein MetalloPhosphatases (PMP), 1 mM MnCl 2 , and 0.4 μL of Lambda Protein Phosphatase (NEB, P0753S) was added to the reaction mix and incubated at 30 °C for 30 minutes.

Techniques: Activation Assay, Recombinant, Incubation, Fluorescence, Negative Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Mass Spectrometry, In Vitro, Western Blot, Mutagenesis, Control

Journal: bioRxiv

Article Title: An automated workflow for quantifying the formation of synuclein aggregates in human dopaminergic neurons

doi: 10.64898/2025.12.19.695578

Figure Lengend Snippet: (A) Overview of pS129-syn aggregate formation assay set-up. (B) Images of phosphorylated synuclein aggregates in SNCA Tri DNs. Two week old DNs are treated with 500 nM α-Syn PFFs or 500 nM α-Syn monomers.Two weeks after the treatment, DNs are fixed and stained for pS129-Syn and TUBB3. Bars = 100 μm. (C) Quantification of pS129-Syn aggregate formation. Total neurite area is identified and a mask for the neurite area is created from the TUBB3 channel image. Spots of pS129-Syn aggregates are identified in the pS129-Syn channel and analyzed within the TUBB3 neurite mask. (D) Confirmation of phosphorylated signal in DNs. DNs from the SNCA Tri line are differentiated for two weeks and then treated with 1 μM PFF for two weeks. After fixation, DNs on coverslips are treated with or without Lambda Protein Phosphatase prior to staining for pS129-Syn and TUBB3. bar = 100 μm. (E and F), Quantification of total pS129-Syn signal (E) and TUBB3 fluorescent signal (F) in SNCA_tri DNs; the data represents the mean±s.e.m. N=3 replicates.

Article Snippet: For samples subjected to phosphatase treatment, cells were fixed and permeabilized and then incubated with 50 units/μL Lambda protein phosphatase (New England Biolabs, cat P0753L) at 30°C for 8 hours before IF staining.

Techniques: Tube Formation Assay, Staining

Journal: bioRxiv

Article Title: Thermo-Sensing Mechanisms of Splicing Control by Nuclear Stress Bodies

doi: 10.1101/2025.10.21.683800

Figure Lengend Snippet: (A) Phos-tag™ SDS-PAGE (upper panel) and standard SDS-PAGE (lower panel) followed by WB of CLK1. Migration of different phosphorylation states is shown on the right. Lambda phosphatase treated sample (+Lambda pp) was used as a reference for dephosphorylated CLK1. DMSO or RIOK2 inhibitors (CQ211, 20 μM; NSC139021, 30 μM) were added during recovery. (B) Schematic of the CLK1 peptide spanning amino acids (aa) 323–343 and bar graph showing the abundance of the phosphorylated peptide across temperature conditions. DMSO or RIOK2 inhibitors were added during recovery as in (A). “1x Phospho” denotes phosphorylation at a single Ser/Thr/Tyr residue. n.d., below the detection limit. Data are presented as mean ± SD (n = 3), with each sample measured twice by mass spectrometry and averaged. **** p < 0.0001, *0.01 < p < 0.05 (Šidák’s multiple comparisons test). (C, E) Subnuclear localization of CLK1 mutants under thermal stress and recovery, visualized by HSATIII RNA-FISH combined with FLAG IF. (C) S/T→A mutant carries alanine substitutions at all serine/threonine residues within CLK1 aa 323-343 (S328A/S330A/S337A/T338A/S341A/T342A). (E) DMSO or CQ211 (10 μM) was added during recovery. Scale bar, 10 μm. (D, F) Quantification of colocalization between CLK1 mutants and nSBs (n = 30) in (C, E). **** p < 0.0001, *0.01 < p < 0.05 (Dunn’s multiple comparisons test). (G) In vitro phosphorylation of RIOK2 and CLK1 assessed by autoradiography with WB validation. (H) Quantification of autoradiographic signal intensities from (G). Each bar corresponds to the lane numbers shown in (G). Data are presented as mean ± SD (n = 3). **** p < 0.0001, ***0.0001 < p < 0.001 (Šidák’s multiple comparisons test).

Article Snippet: For Lambda protein phosphatase treatment, cells expressing FLAG-tagged CLK1 were washed with PBS and lysed in NEBuffer for PMP (NEB) containing 0.1% NP-40.

Techniques: SDS Page, Migration, Phospho-proteomics, Residue, Mass Spectrometry, Mutagenesis, In Vitro, Autoradiography, Biomarker Discovery