pcmv5-smarcc2 (baf170)-flag  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: DNA stimulates the deacetylase SIRT6 to mono-ADP-ribosylate proteins with histidine repeats

doi: 10.1016/j.jbc.2025.108532

Figure Lengend Snippet: SIRT6 mARylates polyHis tract-containing proteins. A – C , immunoblot analyses of SIRT6 (2 μM) mARylation reactions with ( A ) MeCP2-WT or MeCP2 polyHis mutant, ( B ) BAF170-WT or BAF170 polyHis mutant, or ( C ) NLK-WT or NLK polyHis mutant as the substrate (all at 2 μM). SIRT6-H133Y is an inactive mutant that was included as a negative control. The reactions in panels A and C were incubated at 37 °C for 2 h and in panel B for 20 min with 1 mM NAD + and ± 1 μM ds601 DNA, n = 2. The immunoblots in ( A–C ) were performed using a poly/mono-ADP-ribose antibody (Cell Signaling Technologies #83732). D , immunoblot analysis of immunoprecipitated HA-YY1-WT or HA-YY1-ΔpolyHis from HEK293T cells expressing the respective YY1 protein and either FLAG-SIRT6-WT or FLAG-SIRT6-H133Y. The anti-ADPr blot was obtained used a mono-ADP-ribose–specific binding reagent (Millipore #MABE-1076), n = 2. mARylate, mono-ADP-ribosylate; mARylation, mono-ADP-ribosylation; polyHis, polyhistidine; SIRT6, sirtuin6.

Article Snippet: SIRT6 was a gift from Cheryl Arrowsmith (Addgene plasmid #41565; http://n2t.net/addgene:41565;RRID:Addgene_41565 ). pFastBac1 Flag Baf170 was a gift from Robert Kingston (Addgene plasmid #1955; http://n2t.net/addgene:1955 ; RRID:Addgene_1955) ( ).

Techniques: Western Blot, Mutagenesis, Negative Control, Incubation, Immunoprecipitation, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: DNA stimulates the deacetylase SIRT6 to mono-ADP-ribosylate proteins with histidine repeats

doi: 10.1016/j.jbc.2025.108532

Figure Lengend Snippet: SIRT6 mARylation activity is activated by binding to DNA ends. A , titration of ds601 in the MeCP2 (2 μM) mARylation assay by SIRT6 (2 μM), n = 2. B , titration of ds601 and ss601 in the MeCP2 (2 μM) mARylation assay by SIRT6 (2 μM), n = 2. Note that there was no SIRT6 in the reaction for the “no DNA” lane. In terms of concentration, 0.8 μg = 0.5 μM ds601, 1.6 μg = 1 μM ds601, and 3.3 μg = 2 μM ds601. C , immunoblot analysis of mARylation by SIRT6 (2 μM) of MeCP2 (2 μM) in the presence of plasmid DNA (pET28a), n = 2. Note that there was SIRT6 in the reaction for the “no DNA” lane. D , immunoblot analysis of mARylation assays of SIRT6-WT or -ΔC (2 μM) with ds601 or WT mononucleosome (1 μM) with BAF170, MeCP2, or NLK (2 μM) as the substrate, n = 2. The BAF170 reactions were incubated for 20 min. E , immunoblot analysis of WT nucleosomes (200 nM) incubated with SIRT6-WT (2 μM), ds601 (1 μM), and 1 mM NAD + at 37 °C for 2 h, n = 2. The poly/mono-ADP-ribose antibody (Cell Signaling Technologies #83732) was used for all the blots shown here. All the mARylation assays were incubated at 37 °C for 2 h unless otherwise noted. mARylate, mono-ADP-ribosylate; mARylation, mono-ADP-ribosylation; polyHis, polyhistidine; SIRT6, sirtuin6; NLK, nemo-like kinase.

Article Snippet: SIRT6 was a gift from Cheryl Arrowsmith (Addgene plasmid #41565; http://n2t.net/addgene:41565;RRID:Addgene_41565 ). pFastBac1 Flag Baf170 was a gift from Robert Kingston (Addgene plasmid #1955; http://n2t.net/addgene:1955 ; RRID:Addgene_1955) ( ).

Techniques: Activity Assay, Binding Assay, Titration, Concentration Assay, Western Blot, Plasmid Preparation, Incubation

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

doi: 10.1016/j.gim.2023.100950

Figure Lengend Snippet: Figure 1 SMARCC2 linear domain structure, distribution of pathogenic variants in the BAF complex and N-terminal variants causing protein loss. A. Linear protein model of SMARCC2 and its domains: an N-terminal module containing a MarR-like helix-turn-helix domain (eggshell) with DNA-binding ability,25 as well as a BRCT domain (orange) with an inserted non-functional chromodomain (red), which have been proposed to mediate protein-protein interactions.25 The SWIRM domain (magenta) mediates protein-protein interaction,34,35 the SANT domain (berry) is the chromatin binding domain of the protein which was proposed to recognize unmodified histone tails,36,37 the dimerization region (purple) and core assembly region (dark blue) are coiled-coil domains involved in the formation of the core BAF complex. The first is necessary for heterodimerization with SMARCC1 and the latter interacts with SMARCD1 and SMARCE1, forming the base of the

Article Snippet: Missense variants in other BAF base complex subunits (File S2 sheet “missense_other_BAF”) were reviewed from published literature reports (Table S2), standardized to fit the model transcript and used to analyze spatial proximity in the BAF complex model. Functional analyses of missense variants FLAG-tagged SMARCC2 was obtained from Addgene (plasmid #19142)27 and variants were introduced using the InFusion HD Cloning Kit (Clontech).

Techniques: Binding Assay, Functional Assay, Protein-Protein interactions

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

doi: 10.1016/j.gim.2023.100950

Figure Lengend Snippet: Figure 2 Facial appearance and representative cMRIs of SMARCC2 individuals. Facial features, facial overlay and images from hands and feet of individuals with LGD (A) and non-truncating (B) SMARCC2 variants. Ind-7 and Ind-8 carrying the missense variant c.230C>T p.(Pro77Leu), which leads to SMARCC2 protein loss, were grouped together with the LGD variant individuals. In addition to other craniofacial anomalies, a triangular face with narrow chin is frequently depicted in photos of both groups. Also note the pronounced coarseness of facial characteristics in individuals with non-truncating variants. C. Neuroimaging characteristics of SMARCC2 individuals and a control subject. Ind-02, Ind-09 and Ind-11 exhibit normal corpus callosum appearance. Corpus callosum hypoplasia (empty arrows) or dysplasia with thinning of the corpus callosum splenium (thick arrows), may or may not be accompanied by anterior commissure hypoplasia/ agenesis (arrowheads) and small inferior cerebellar vermis (thin arrows).

Article Snippet: Missense variants in other BAF base complex subunits (File S2 sheet “missense_other_BAF”) were reviewed from published literature reports (Table S2), standardized to fit the model transcript and used to analyze spatial proximity in the BAF complex model. Functional analyses of missense variants FLAG-tagged SMARCC2 was obtained from Addgene (plasmid #19142)27 and variants were introduced using the InFusion HD Cloning Kit (Clontech).

Techniques: Variant Assay, Control

Journal: bioRxiv

Article Title: Anoctamin 10/TMEM16K mediates convergent extension and tubulogenesis during notochord formation in the early chordate Ciona intestinalis

doi: 10.1101/2023.01.20.524945

Figure Lengend Snippet: (A-B) Montage of two time-lapse movies from different embryos expressing Ciinte . Brac>GCaMP6s during notochord cell intercalation. White arrowheads point to notochord cells with changing fluorescence intensity in response to changes in Ca 2+ concentration (Supplementary Movies 4-8). (C) Three example Ca 2+ traces from notochord cells. (D-G) Quantification of features from negative control and Ano10 CRISPR Ca 2+ transients during convergent extension. Violin plots for (D) amplitude (E) rising slope, (F) falling slope (G) duration. The red line indicates the median and green lines indicate the quartiles. (H, I) Montage of two-time lapse movies from different embryos expressing Ciinte . Brac>GCaMP6s during lumen formation and extension. (J-M) Violin plots quantifying (J) amplitude (K) rising slope, (L) falling slope and (M) duration of Ca 2+ transients from control and Ano10 CRISPR embryos during tubulogenesis. For statistical analysis of data shown in panels ((D-G; J-M) we performed Mann-Whitney tests (see also Supplementary Table 4). (K,L) Quantification of Red/Green ratio of CAMPARI Ca 2+ integrator before (pre) and after (post) photoconversion at the end of CE (K) and tubulogenesis (L). For statistical analysis we performed a Kurskal-Wallis test followed by Dunn’s multiple comparisons test (see also Supplementary Table 4)

Article Snippet: For the middle position we used hCD4GFP, GCaMP6s, nls::Cas9::nls (subcloned from Eef1a-1955/- 1>nls::Cas9::nls a gift from Lionel Christiaen, Addgene plasmid # 59987 ; http://n2t.net/addgene:59987 ; RRID:Addgene_59987) , EB3-mNeonGreen a gift from Dorus Gadella (Addgene plasmid # 98881 ; http://n2t.net/addgene:98881 ; RRID:Addgene_98881), TdTomato-LifeAct tdTomato-Lifeact-7 was a gift from Michael Davidson (Addgene plasmid # 54528 ; http://n2t.net/addgene:54528 ; RRID:Addgene_54528).

Techniques: Expressing, Fluorescence, Concentration Assay, Negative Control, CRISPR, Control, MANN-WHITNEY