Journal: bioRxiv

Article Title: Direct binding of a fungal effector by the wheat RWT4 tandem kinase activates defense

doi: 10.1101/2024.04.30.591956

Figure Lengend Snippet: (A) RWT4 Ca domain organization. Kinase domain: 1-478 amino acid, green. Pseudokinase domain: 540-915 amino acid, blue. Linker: 478-540 amino acid, grey. Predicted kinase active sites: K217 and K327. Dash: C-terminal tail. (B) Purity of recombinant proteins isolated from E. coli. His-MBP-RWT4 Ca , kinase domain (His-MBP-KD) pseudokinase domain (His-MBP-PKD), and its kinase dead variant (RWT4 Ca_K217R/K327R ) are shown by Coomassie brilliant blue (CBB) stained SDS-PAGE and corresponding western blot using anti-His. (C) Autophosphorylation activity of His-MBP-RWT4 Ca . Autophosphorylation status of the His-MBP-RWT4 Ca proteins were assayed by western blot using anti-phospho-Threonine/Tyrosine antibody (α-pThr/Tyr) with or without lambda protein phosphatase incubation prior to separating by SDS-PAGE. Anti-His western blotting (α-His) demonstrates equal loading of the His-MBP-RWT4 Ca proteins. (D) Active protein phosphorylation was measured by incubating the His-MBP-CaRWT4 proteins with [γ -32P ]ATP before separating by SDS-PAGE and visualizing by autoradiography. (E) RWT4 Ca -AvrPWT4 triggered cell death requires RWT4 Ca kinase activity. Rwt4 Ca and the kinase dead variant ( Rwt4 Ca _K217R/K327R ) were co-transfected with AvrPwt4 into rice protoplasts for cell viability assays. Five independent protoplast transfection events were used as biological replicates and Mean±SEM are presented. Treatment differences were analyzed using one-way ANOVA with post-hoc Tukey HSD, p<0.05. (F) RWT4 Ca specifically trans-phosphorylates AvrPWT4. Transphosphorylation activity was assayed by mixing His-MBP-RWT4 Ca and AvrPWT4 at room temperature for 20 min before incubated with [γ- 32 P]ATP. Phosphorylation of the substrate proteins (AvrPWT4 or MyBP) were visualized via autoradiography. (G) Phosphorylated peptides were mapped to the sequence of AvrPWT4 for functional analysis. The phosphorylated peptide YIITDDG was identified from LC-MS/MS with the phosphorylation site T45. Rwt4 Ca was co-transfected with either wild-type AvrPwt4 or the phosphorylation null mutant AvrPwt4 T45A to rice protoplasts. Luminescence (cd/m 2 ) was measured 16 h after protoplast transfection as an indicator of cell viability. Six independent protoplast transfection events were used as biological replicates. Mean±SEM are presented. Differences between treatments were analyzed using one-way ANOVA with post-hoc Tukey HSD, p<0.05.

Article Snippet: For phosphatase treatments, 2µg of recombinant protein was mixed with 400U lambda protein phosphatase (NEB, #P0753S) at 30°C for 30 min before separated by an SDS-PAGE.

Techniques: Recombinant, Isolation, Variant Assay, Staining, SDS Page, Western Blot, Activity Assay, Incubation, Autoradiography, Transfection, Sequencing, Functional Assay, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: Science (New York, N.Y.)

Article Title: In silico protein interaction screening uncovers DONSON’s role in replication initiation

doi: 10.1126/science.adi3448

Figure Lengend Snippet: (A-B) Models of CMG assembly in budding yeast and vertebrates. (C) Relative DNA replication efficiency in the indicated egg extracts. Because depletion of DONSON co-depletes roughly half of the 0.5 to 1 µM endogenous CDK2-Cyclin E, DONSON-depleted (ΔDONSON) extracts but not mock-depleted extracts were supplemented with 0.3 µM recombinant human CDK2-Cyclin E1. Recombinant DONSON (rDONSON, ) was added where indicated. Datapoints, n=3 experiments. Mean±SD. A representative western blot of total protein levels in these reactions is shown in . (D) Plasmid DNA was incubated in the indicated egg extracts. At the specified times following NPE addition, chromatin was recovered and blotted for the indicated proteins. DONS, DONSON; Gem, geminin; CDKi, p27 Kip . A representative western blot of total protein levels in these reactions is shown in . (E) Plasmid DNA was incubated in extracts depleted of DONSON and/or RPA. At the specified times following NPE addition, chromatin was recovered and blotted for the indicated proteins. Western blot of total protein levels in these reactions is shown in . (F) In the presence of the indicated inhibitors of replication initiation, plasmid DNA was recovered 10 minutes after NPE addition and blotted for the indicated proteins. DDKi, PHA-767491.

Article Snippet: In , extracts were supplemented with 50 μg/mL recombinant GST-p27 Kip (“CDKi”) or 20 U/μL Lambda protein phosphatase (New England BioLabs P0753) and treated for 30 mins at room temperature prior to immunoprecipitation.

Techniques: Recombinant, Western Blot, Plasmid Preparation, Incubation

Journal: Science (New York, N.Y.)

Article Title: In silico protein interaction screening uncovers DONSON’s role in replication initiation

doi: 10.1126/science.adi3448

Figure Lengend Snippet: (A) Recombinant FLAG-tagged DONSON (rFLAG-DONSON) was added to non-replicating nucleoplasmic egg extract (NPE), recovered, and blotted for the indicated proteins alongside the input extract. (B) Endogenous GINS was immunoprecipitated from NPE using PSF3 antibody and blotted for the indicated proteins. (C) rFLAG-DONSON proteins containing specified mutations ( and ) were added to NPE, recovered, and blotted as indicated. Red and blue bars show missing pre-LC components. The images are part of the same western blot, which was cropped to remove irrelevant information between lanes 5 and 6. (D) The effects of different DONSON mutants are depicted in the context of the AF-M-modeled pre-LC (as in ). Mutations are indicated as red Xs. (E) The indicated rFLAG-DONSON proteins were added to NPE treated with buffer, p27 Kip (CDKi), or λ phosphatase. DONSON was recovered and blotted for the indicated proteins. Total extract was also blotted for MCM4 to show λ phosphatase activity.

Article Snippet: In , extracts were supplemented with 50 μg/mL recombinant GST-p27 Kip (“CDKi”) or 20 U/μL Lambda protein phosphatase (New England BioLabs P0753) and treated for 30 mins at room temperature prior to immunoprecipitation.

Techniques: Recombinant, Immunoprecipitation, Western Blot, Activity Assay

Journal: Nucleic Acids Research

Article Title: CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling

doi: 10.1093/nar/gkad298

Figure Lengend Snippet: CSNK2B interacts with IRF1 and regulates its antiviral activity independently of phosphorylation. ( A ) 293FT cell lysates expressing IRF1-FLAG or empty vector was immunoprecipitated with anti-FLAG M2 antibody. Proteins eluted from the precipitates were subjected to an SDS-PAGE followed by western blotting with specific antibodies against CK2 components. ( B ) Pull-down analysis showing direct interaction between purified IRF1-FLAG and recombinant human CSNK2B (rCSNK2B) proteins. ( C ) Phos-tag SDS-PAGE of FLAG-tagged IRF1. PH5CH8 cell lysates were subjected to immunoblotting before (−) and after (+) digestion with lambda protein phosphatase (λ PPase). An arrowhead shows phospho-IRF1. ( D , E ) Phos-tag gel analysis of ectopically expressed IRF1-FLAG in 293FT cells ( D ) or endogenously expressed IRF1 in PH5CH8 cells ( E ). Means ± S.D. of values for abundance of phospho-IRF1 relative to non-phosphorylated IRF1 are shown on right ( n = 3). ( F ) PH5CH8 cells were transfected with siRNAs targeting CK components and infected 48 h later with HAV at an m.o.i. of 10. Percentage of HAV RNA levels relative to non-target control siRNA was determined 4 d.p.i. by RT-qPCR. Immunoblots showing depletion of catalytic CK2 subunits are shown on right. ** P < 0.01 versus control ( n = 3 or 4, one-way ANOVA with Dunnett's multiple comparisons test). ( G ) Immunoblots showing CSNK2B protein abundance in Huh-7.5 cells treated with 10 μM CX-4945 for 24 h. ( H ) Effects of CX-4945 on replication of HAV/NLuc (18f/NLuc) in Huh-7.5 cells and cell viability. ** P < 0.01, *** P < 0.0001 versus control ( n = 3, one-way ANOVA with Bonferroni's multiple comparisons test). ( I ) Effects of CX-4945 on HAV replication and PLAAT4 expression in IRF1 -depleted and control Huh-7.5 cells. ** P < 0.01, *** P < 0.0001 ( n = 3, two-way ANOVA with Sidak's multiple comparisons test). ( J ) NLuc reporter analysis of Huh-7.5 cells expressing NLuc reporter (pNL-4×IRF1). Cells were transfected with indicated siRNAs for 48 h, and relative NLuc values secreted at 48–72 h post-transfection are shown. Immunoblots showing depletion of each siRNA target are shown on right. * P < 0.05, ** P < 0.01 versus control ( n = 4, one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Lambda protein phosphatase was obtained from Bio Academia. miR-122 mimics were synthesized by Dharmacon and transfected by electroporation as miRNA/miRNA* duplexes as described ( ).

Techniques: Activity Assay, Phospho-proteomics, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Purification, Recombinant, Transfection, Infection, Control, Quantitative RT-PCR, Quantitative Proteomics

Journal: Nucleic Acids Research

Article Title: CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling

doi: 10.1093/nar/gkad298

Figure Lengend Snippet: CSNK2B regulates transcription and phosphorylation of AFAP1 that lowers permissiveness to flavivirus replication. ( A ) Immunoblots of AFAP1 and GAPDH as loading control in lysates of PH5CH8 cells transfected with indicated siRNAs, with and without IFNγ stimulation. ( B ) Immunoblots of AFAP1, IRF1 and GAPDH in lysates of PH5CH8 cells depleted of either (or both) CSNK2B and IRF1. Quantitation of AFAP1 protein abundance is shown on the right. ** P < 0.01, *** P < 0.0001 ( n = 3, one-way ANOVA with Dunnett's multiple comparisons test). ( C ) Immunoblots of AFAP1 and GAPDH in lysates of PH5CH8 cells treated with either lambda protein phosphatase (λ PPase, left panels) or indicated concentrations of CX-4945 (right panels). ( D ) Immunoblots of AFAP1 and Src in lysates of PH5CH8 cells treated with 10 μM CX-4945. ( E ) Immunoblots of PH5CH8 cell lysates transfected with indicated siRNAs targeting AFAP1 (left panels) or CSNK2B (right panels). ( F ) Scheme of CSNK2B-regulated AFAP1 signaling cascades that lead to Src activation. ( G ) Immunoblots of PH5CH8 cell lysates transfected with indicated siRNAs (top). DENV RNA levels were determined at 48 h p.i. (bottom). * P < 0.05 ( n = 3, two-tailed Student's t -test). ( H ) Immunoblots of AFAP1 in lysates of PH5CH8 versus Huh-7.5 cells stably transduced with AFAP1 (top). Huh-7.5 cells stably expressing AFAP1 or vector control were challenged with DENV/NLuc (bottom). NLuc activities at the indicated time points post-infection are shown. ** P < 0.01 ( n = 3, two-way ANOVA with Sidak's multiple comparisons test). ( I ) PH5CH8 cells were transfected with indicated siRNAs and infected 48 h later with dengue virus (DENV) at an m.o.i. of 0.1. CX-4945 (5 μM) was added 2 h post-infection. Infectious titers were determined 48 h p.i. by focus formation assays. FFU, focus forming units. ** P < 0.01, *** P < 0.0001 versus control ( n = 3, one-way ANOVA with Dunnett's multiple comparisons test). ( J ) PH5CH8 cells were infected with DENV or Zika virus (ZIKV) as in (I) and viral RNA levels determined by RT-qPCR. ** P < 0.01 ( n = 3, two-tailed Student's t -test).

Article Snippet: Lambda protein phosphatase was obtained from Bio Academia. miR-122 mimics were synthesized by Dharmacon and transfected by electroporation as miRNA/miRNA* duplexes as described ( ).

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Quantitation Assay, Quantitative Proteomics, Activation Assay, Two Tailed Test, Stable Transfection, Transduction, Expressing, Plasmid Preparation, Infection, Virus, Quantitative RT-PCR

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet:

Article Snippet: Recombinant Lambda Protein Phosphatase , NEB , Cat# P0753.

Techniques: Transduction, Binding Assay, Recombinant, Staining, Sonication, Mass Spectrometry, Protease Inhibitor, Labeling, Transfection, Polymer, Live Cell Imaging, Protein Purification, CRISPR, Imaging, Plasmid Preparation, Software, Western Blot