flag peptide  (MedChemExpress)

Journal: Science Advances

Article Title: YAP/TAZ-VGLL3 governs adipocyte fate via epigenetic reprogramming of PPARγ and its target enhancers

doi: 10.1126/sciadv.aea7235

Figure Lengend Snippet: ( A ) The 3D structure of human TAZ (UniProt accession Q9GZV5 ) predicted by AlphaFold is shown on the left. The domain organization of human TAZ is shown on the right: TEAD binding domain (TBD), WW domain, coiled-coil (CC) domain, transactivation domain (TAD), and PDZ binding motif. Serine-to-alanine (SA) mutation sites for constitutively active mutants (red circles) and a TEAD-binding–deficient mutant (blue circle). ( B and C ) Streptavidin pull-down assay for human embryonic kidney (HEK) 293T cells showing the interaction of green fluorescent protein (GFP)–tagged PPARγ2 (B) or endogenous TEADs and LATS1 (C) with SFB (S protein–FLAG–streptavidin binding peptide)–tagged TAZ mutants. ( D to F ) C3H10T1/2 cells stably expressing doxycycline-inducible 2xHA-tagged TAZ mutants were subjected to immunoblot analysis (D), RT-qPCR analysis ( n = 3) (E), and lipid staining with oil red O (F). Scale bars, 200 μm (F). ( G ) MA plot showing differential H3K27ac ChIP-seq signals between control and TAZ2SA-expressing C3H10T1/2 cells (left) and heatmaps showing H3K27ac ChIP-seq signals for cells expressing the indicated TAZ2SA mutants (right). Significantly changed regions (|fold change| > 2 and adj. P value of <0.05) in TAZ2SA-expressing versus control cells are colored red (fold change > 2) or blue (fold change < −2). ( H ) Genome browser views of the indicated loci showing the coverage of the H3K27ac ChIP-seq signal in differentiated C3H10T1/2 cells expressing the TAZ mutants. ( I ) Heatmap of the H3K27ac ChIP-seq signals from C3H10T1/2 cells expressing indicated TAZ mutants, aligned with PPARγ ChIP-seq peaks (D6). Data in bar graph (E) are means ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For streptavidin-mediated pull-down assay of S protein–FLAG–streptavidin binding peptide (SFB)–tagged proteins, cleared cell lysates (1 mg of protein in 1 ml) were incubated for 2 hours at 4°C with 20 μl of Pierce High Capacity Streptavidin Agarose (20359, Thermo Fisher Scientific), and the beads were then washed three times with lysis buffer and boiled with Laemmli sample buffer for immunoblot analysis.

Techniques: Binding Assay, Mutagenesis, Pull Down Assay, Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR, Staining, ChIP-sequencing, Control

Journal: Science Advances

Article Title: YAP/TAZ-VGLL3 governs adipocyte fate via epigenetic reprogramming of PPARγ and its target enhancers

doi: 10.1126/sciadv.aea7235

Figure Lengend Snippet: ( A ) Scatter plot showing correlations between RNA fold change and ATAC gene activity change in adipocyte-related cells (LAKO versus control; sn sequencing). Color indicates the maximum percentage of cells expressing each gene. ( B ) Vgll3 expression in iWAT snRNA-seq. Dediff., Dedifferentiated adipocytes. ( C ) Genomic browser view of Vgll3 locus with the indicated sequencing data. ( D ) Mouse adipose tissue RNA-seq data ( GSE138911 ) showing Vgll3 expression [fragments per million mapped reads (FPM)] in high-fat diet (HFD) or normal chow (NC)–fed adipocyte-specific YAP/TAZ KO (YTKO) mice ( Yap1 fl/fl ; Wwtr1 fl/fl ; Adipoq-Cre ). ( E ) Human visceral adipose tissue RNA data from the GTEx consortium showing the correlation between WWTR1 and VGLL3 expression (right). ( F ) C3H10T1/2 cells expressing doxycycline (Dox)–inducible HA-TAZ2SA were treated with 2 μM VT-104 for 36 hours and subjected to RT-qPCR. ( G ) C3H10T1/2 cells (D6) expressing Dox-inducible Myc-Vgll3 were analyzed by RT-qPCR ( n = 3) and immunoblot. ( H ) Oil red O staining of Dox-inducible Vgll3 or Vgll3ΔTDU-expressing cells. Scale bars, 200 μm. ( I ) Vgll3 KO C3H10T1/2 cells expressing Dox-inducible HA-TAZ2SA and its parental cells (Con) were analyzed by RT-qPCR and immunoblot. ( J ) Genomic view of Pparg (left) and Fabp4 (right) regions with the indicated ChIP-seq data from TAZ mutant–expressing cells. ( K ) H3K27ac peak heatmap aligned with PPARγ ChIP-seq peaks (D6). ( L ) Streptavidin pull-down assay of HEK293T cells cotransfected with HA-tagged histone deacetylase 3 (HDAC3) and SFB-tagged VGLL3. ( M ) C3H10T1/2 cells with inducible Vgll3 expression (TRE-Vgll3) and Hdac3 [short hairpin Hdac3 (shHdac3)] or Ncor1 (shNcor1) knockdown or parental control (−) were treated with adipogenic cocktails and Dox or vehicle control (48 hours) and subjected to RT-qPCR. ( N ) A proposed model for the role of TAZ in adipocyte differentiation and dedifferentiation. Data in bar graphs (D, E, and G) are means ± SEM and analyzed by the unpaired t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For streptavidin-mediated pull-down assay of S protein–FLAG–streptavidin binding peptide (SFB)–tagged proteins, cleared cell lysates (1 mg of protein in 1 ml) were incubated for 2 hours at 4°C with 20 μl of Pierce High Capacity Streptavidin Agarose (20359, Thermo Fisher Scientific), and the beads were then washed three times with lysis buffer and boiled with Laemmli sample buffer for immunoblot analysis.

Techniques: Activity Assay, Control, Sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Staining, ChIP-sequencing, Mutagenesis, Pull Down Assay, Histone Deacetylase Assay, Knockdown

Journal: Nature Communications

Article Title: Shigella flexneri evades septin-mediated cell-autonomous immunity via protein ADP-riboxanation

doi: 10.1038/s41467-026-68425-0

Figure Lengend Snippet: a Coomassie blue-stained purified 3×FLAG-SEPT9 from 293T cells co-transfected with GFP-OspC3-WT or GFP-OspC3-EH/AA prior to MS analyses. Right: ClustalW2 multiple sequence alignment of SEPT3 subgroup members. Key salt-bridge residues at the NC-interface are marked with yellow circles. ADP-riboxanation sites are highlighted in red; asterisks indicate conserved residues. The six arginine residues labeled in black were identified after the initial four sites were mutated to lysine (4RK). b Extracted ion chromatograms of an unmodified peptide, an Arg561-containing peptide and its ADP-riboxanated form from immunoprecipitated SEPT9 in ( a ). c Tandem mass spectrum of the ADP-riboxanated Arg561-containing peptide acquired under collision-induced dissociation (CID). d , e Validation of MS-identified sites by site-directed mutagenesis. 293T cells co-transfected with GFP-OspC3 and FLAG-SEPT9 (WT or variants) were analyzed by IP and immunoblotting. 10RK refers to substitution of all ten modified arginine residues to lysine. f Prokaryotic co-expression. GST-SEPT9 was expressed in E. coli alone or with calmodulin (CaM) and OspC3 as indicated. Purified GST-SEPT9 was blotted to detect ADP-riboxanation. g In vitro reconstitution. Purified recombinant proteins were incubated (37 °C, 1 h) and analyzed by Coomassie staining and Western blotting. The effects of NAD + , CaM, and calcium ions were tested. h ADP-riboxanation during infection. HeLa cells transfected with FLAG-SEPT2, SEPT6, SEPT7, or SEPT9 were infected with S. flexneri (MOI = 50, 2 h). Anti-FLAG immunoprecipitates were analyzed by immunoblotting. i OspC-dependent modification of Arg561. HeLa cells expressing FLAG-SEPT9 (WT or R561 mutants) were infected with indicated S. flexneri strains (MOI = 50, 2 h). Modification was assessed by anti-FLAG IP and immunoblotting. UI, uninfected. j Endogenous SEPT9 modification. Analyzed as in ( i ), but immunoprecipitated with an anti-SEPT9 antibody. For ( a , d – j ), experiments were repeated three times with similar results.

Article Snippet: The clarified lysates were incubated with pre-washed anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) at 4 °C with gentle rotation for 4 h. The beads were washed four times with the lysis buffer, and bound proteins were eluted with FLAG peptides (MedChemExpress, HY-P0319A) and run on SDS-PAGE followed by either Western blot or LC-MS analyses.

Techniques: Staining, Purification, Transfection, Sequencing, Labeling, Immunoprecipitation, Biomarker Discovery, Mutagenesis, Western Blot, Modification, Expressing, In Vitro, Recombinant, Incubation, Infection

Journal: Nature Communications

Article Title: Shigella flexneri evades septin-mediated cell-autonomous immunity via protein ADP-riboxanation

doi: 10.1038/s41467-026-68425-0

Figure Lengend Snippet: a Quantitative proteomic analysis of the SEPT9 interactome upon ADP-riboxanation. 293T cells co-expressing 3×FLAG-SEPT9 with GFP-OspC3-WT or -EH/AA were analyzed by anti-FLAG IP and quantitative MS. Volcano plot shows fold changes (x-axis) vs. statistical significance (y-axis) of interacting proteins. Data represent four biological replicates. Statistical analysis: two-sided paired moderated t -test (limma). b , c Impaired septin subunit interactions. 293T cells co-transfected with EGFP-OspC3 (WT or EH/AA), FLAG-SEPT9, and HA-SEPT2 or HA-SEPT6 were analyzed by anti-FLAG IP and immunoblotting. Quantification of interaction strength is shown below blots. d Network analysis. SEPT9-associated proteins are grouped into functional clusters. Nodes are color-coded by interaction strength (log2 fold changes). e Interaction with endogenous septins. 293T cells co-transfected with EGFP-OspC3 (WT or EH/AA) and FLAG-SEPT9 (WT, 4RK, or 10RK) were analyzed by anti-FLAG IP and immunoblotting for endogenous SEPT2, SEPT6, and SEPT7. Quantification is shown below blots. f In vitro pull-down assay. Recombinant GST-Strep-tag II-SEPT9 (WT, ADP-riboxanated, 4RK, or 10RK) and GST-SEPT7 individually purified from E. coli were incubated together. Strep-Tactin pull-down samples were analyzed by immunoblotting. Quantification is shown below blots. For ( b , c , e , f ), experiments were repeated three times with similar results.

Article Snippet: The clarified lysates were incubated with pre-washed anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) at 4 °C with gentle rotation for 4 h. The beads were washed four times with the lysis buffer, and bound proteins were eluted with FLAG peptides (MedChemExpress, HY-P0319A) and run on SDS-PAGE followed by either Western blot or LC-MS analyses.

Techniques: Expressing, Transfection, Western Blot, Functional Assay, In Vitro, Pull Down Assay, Recombinant, Strep-tag, Purification, Incubation