anti flag primary antibody  (Cell Signaling Technology Inc)

Journal: Materials Today Bio

Article Title: Inhalable engineered probiotic outer membrane vesicles co-expressing multiple mpox antigens induce potent specific systemic and mucosal immune responses

doi: 10.1016/j.mtbio.2026.103089

Figure Lengend Snippet: Design, Preparation, and Characterization of ABM@OMV. ( A ) Schematic illustrating the preparation of ABM@OMV. The pThioHisA plasmid, encoding Trx-A9R and ClyA-B6R-M1R fusion proteins, was transformed into Δ lpxM Escherichia coli Nissle 1917 (ΔEcN). OMVs were harvested via ultracentrifugation. ( B ) Bacterial lysates analyzed by SDS-PAGE and stained with Coomassie blue. Lanes: Marker; uninduced ΔEcN; ΔEcN + IPTG; uninduced BMΔEcN; BMΔEcN + IPTG; uninduced ABMΔEcN; ABMΔEcN + IPTG. Black boxes indicate ClyA-B6R-M1R and Trx-A9R fusion proteins. ( C ) Western blot analysis of proteins of interest expressed in ABMΔECN bacteria. Anti-His tag antibody detected Trx-A9R; anti-Flag tag antibody detected ClyA-B6R-M1R. Lane order is the same as in B . ( D ) Dynamic light scattering (DLS) size distribution profiles of Δ lpxM OMVs, BM@OMV, and ABM@OMV. ( E ) Zeta potential measurements of Δ lpxM OMVs, BM@OMV, and ABM@OMV ( n = 3). ( F ) Transmission electron microscopy (TEM) images of Δ lpxM OMVs, BM@OMV, and ABM@OMV. Scale bar: 100 nm. ( G ) Western blot analysis of proteins of interest expressed in ABM@OMV. Lanes: Δ lpxM OMVs, BM@OMV, ABM@OMV. ( H ) Endotoxin levels in Δ lpxM EcN-derived OMV and wild-type EcN-derived OMV, as measured by Limulus amebocyte lysate (LAL) assay ( n = 3). ( I ) Representative western blot showing the stability of proteins of interest in Δ lpxM OMVs, BM@OMV, and ABM@OMV following storage at 4 °C (left) or −80 °C (right) for the indicated time periods for the indicated time periods. ( J, K ) Changes in particle size ( J ) and zeta potential ( K ) of OMVs stored at 4 °C and −80 °C ( n = 3). All data were analyzed with GraphPad Prism 8 and are presented as mean ± SD. For panel H , statistical significance between two groups was determined by an unpaired two-tailed t -test. ∗∗∗ P < 0.001.

Article Snippet: The membrane was blocked with 5% BSA (BSA0020, Biosharp, China), then incubated with anti-His tag antibody (1:10,000, HY-P809476X, Med ChemExpress, USA) and anti-FLAG antibody (1:10,000, HY- P80111 , Med ChemExpress, USA), respectively for A9R and B6R-M1R, followed by HRP-conjugated secondary antibodies.

Techniques: Plasmid Preparation, Transformation Assay, SDS Page, Staining, Marker, Western Blot, Bacteria, FLAG-tag, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Derivative Assay, LAL Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Mitochondrial Localization of Cx26 Promotes Colorectal Cancer Malignancy and Exposes a Fatal Vulnerability to Drug Targeting

doi: 10.1002/advs.78117

Figure Lengend Snippet: PIMT promotes mitochondrial translocation of Cx26 in highly malignant CRC cells. (A) HEK293T cells were transfected with Cx26‐HA and PIMT‐Flag, and then immunoprecipitated with IgG as a control or anti‐Flag antibody or anti‐HA antibody, followed by western blot for the indicated proteins. (B) Analyze the correlation between the mRNA levels of PIMT and Cx26 in colorectal cancer cell lines through the TCGA database. (C) Immunoblot of PIMT and Tubulin in SW480 and SW620 cells. Tubulin was used as a loading control. *** p < 0.001 vs . SW480; by unpaired student's t ‐test (mean ± SEM). (D) Lysates of SW480 and SW620 cells were co‐immunoprecipitated with anti‐Cx26 or anti‐PIMT antibodies, and then PIMT and Cx26 protein levels were detected by immunoblot. IgG was used as a loading control. (E) Immunofluorescence of Cx26 (green) and PIMT (red) in SW480 and SW620 cells. Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (F) SW480 cells were treated with TGF‐β for 0, 24, and 48 h; cell lysates were collected for co‐immunoprecipitation with anti‐Cx26 antibody. IgG was used as a loading control. (G) Western blot of the protein level of Cx26 in the cytoplasm and plasma membrane in SW620 cells transfected with shRNA‐NC or shRNA‐PIMT. Na, K‐ATPase and Tubulin were measured as loading control of membrane and cytoplasm, respectively. (H) Immunofluorescence of Cx26 (green) in SW620 cells transfected with or without shRNA‐PIMT. Mitochondria were stained with MitoTracker Red (red). Nuclei were stained with DAPI (blue). Scale bar, 50 µm. (I) Immunoblot of Cx26, PIMT, COX4, and Tubulin in the cytoplasmic and mitochondria‐enriched fractions of SW620 cells transfected with or without shRNA‐PIMT. Tubulin and COX4 were used as markers and loading controls for the cytoplasmic and mitochondria‐enriched fractions, respectively. *** p < 0.001 vs . SW620 cells transfected without shRNA‐PIMT; by unpaired student's t ‐test (mean ± SEM).

Article Snippet: Anti‐Cx26 antibody, anti‐Calnexin antibody, and anti‐Flag antibody were purchased from Proteintech Group (CHI, USA).

Techniques: Translocation Assay, Transfection, Immunoprecipitation, Control, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Clinical Proteomics, Membrane, shRNA

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: PRIMA‐1 Met disrupts p53 R273H ‐PDPK1 interaction. (A) U‐251MG cells were treated with the indicated doses of PRIMA‐1 Met for 24 h. Lysates were immunoprecipitated with anti‐p53 antibody and analyzed by western blot using the indicated antibodies (representative of n = 2 independent experiments). Additional validation is provided in Fig. . (B) Proximity ligation assay (PLA) in U‐251MG cells. Cells transfected with FLAG‐PDPK1 were fixed, permeabilized, and stained using anti‐p53 and anti‐FLAG antibodies for PLA. Confocal microscopy images show nuclei (DAPI, blue) and green fluorescence signals indicating positive PLA interactions (representative of n = 3 independent experiments). Scale bar = 50 μm; applies to all the images in the panel. (C) FRET‐FLIM analysis of NCI‐H1299 cells transfected with the indicated plasmids. Gray circles represent individual cells (number of cells = 171, 245, 115 from left to right). Blue horizontal lines and gray error bars represent the mean homodimerization fraction ± SEM for each group (representative of n = 3 independent experiments). Statistical analysis was performed using a one‐way ANOVA with post hoc Tukey's test. **** P value < 0.0001. (D) U‐251MG cells were treated for 24 h in DMEM containing 0.1% FBS with DMSO, TNFα, PRIMA‐1 Met , or a combination of TNFα and PRIMA‐1 Met . Lysates were analyzed by western blot using the indicated antibodies (representative of n = 2 independent experiments). (E) A schematic model illustrating the p53 R273H ‐PDPK1 interaction contributing to increased AKT phosphorylation. The p53 R273H mutant disrupts the inactive PDPK1 homodimer, resulting in the formation of active PDPK1 monomers that promote AKT phosphorylation. Treatment with PRIMA‐1 Met , which has been reported to restore p53 R273H to a wild‐type‐like conformation, disrupts the p53 R273H ‐PDPK1 interaction and inhibits this gain‐of‐function effect of p53 R273H .

Article Snippet: Briefly, cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126) and rabbit anti‐FLAG (Proteintech, #80010‐1‐RR; probing for FLAG‐tagged PDPK1) primary antibodies.

Techniques: Immunoprecipitation, Western Blot, Biomarker Discovery, Proximity Ligation Assay, Transfection, Staining, Confocal, Microscopy, Fluorescence, Analysis, Phospho-proteomics, Mutagenesis

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: Mutant p53 R273H binds to PDPK1. (A) A schematic workflow of the screening approach. Lysates from U‐251MG cells expressing p53 R273H ‐FLAG were immunoprecipitated with anti‐FLAG antibody, and the immunoprecipitated proteins were analyzed by mass spectrometry. (B) KEGG pathway analysis of the 151 p53 R273H ‐interacting proteins using DAVID (Database for Annotation, Visualization and Integrated Discovery). The top 10 enriched pathways are shown. (C and D) Co‐immunoprecipitation of exogenous p53 R273H and FLAG‐PDPK1. Lysates of NCI‐H1299 cells transfected with the indicated plasmids were immunoprecipitated with anti‐p53 (C) or anti‐FLAG (D) antibody and analyzed by western blot using the indicated antibodies (both panels are representative of n = 3 independent experiments). (E) GST pulldown assay. Purified 6 × His‐PDPK1 was incubated with GST or GST‐p53 R273H proteins immobilized on glutathione agarose beads and analyzed by western blot using the indicated antibodies (representative of n = 4 independent experiments). (F) Co‐immunoprecipitation of endogenous p53 R273H and PDPK1. Lysates of U‐251MG cells transduced with shControl‐ or sh TP53 ‐expressing retroviruses were immunoprecipitated with anti‐p53 antibody and analyzed by western blot using the indicated antibodies (representative of n = 5 independent experiments). (G) Proximity ligation assay (PLA) in U‐251MG cells. Cells transfected with FLAG‐PDPK1 were fixed, permeabilized, and stained using anti‐p53 and anti‐FLAG antibodies for PLA. Confocal microscopy images show FLAG‐PDPK1 (red), p53 R273H (gray), nuclei (DAPI, blue), and green fluorescence signals indicating positive PLA interactions (representative of n = 3 independent experiments). Scale bar = 50 μm (applies to all the images in the panel).

Article Snippet: Briefly, cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126) and rabbit anti‐FLAG (Proteintech, #80010‐1‐RR; probing for FLAG‐tagged PDPK1) primary antibodies.

Techniques: Mutagenesis, Screening, Expressing, Immunoprecipitation, Mass Spectrometry, Analysis, Co-Immunoprecipitation Assay, Transfection, Western Blot, GST Pulldown Assay, Purification, Incubation, Transduction, Proximity Ligation Assay, Staining, Confocal, Microscopy, Fluorescence

Journal: Molecular Oncology

Article Title: Mutant p53 R273H disrupts PDPK1 homodimerization and contributes to PDPK1 activation

doi: 10.1002/1878-0261.70341

Figure Lengend Snippet: F270A mutation in p53 R273H disrupts PDPK1 binding. (A) Lysates of NCI‐H1299 cells transfected with the indicated plasmids were immunoprecipitated with anti‐FLAG antibody and analyzed by western blot using the indicated antibodies (representative of n = 2 independent experiments). Additional validation is provided in Fig. . (B) Molecular Dynamics (MD) simulations on the p53 DNA binding domain (DBD) structural changes following point mutations at specific amino acid positions. Amino acid positions 270 (in orange), 273 (in blue), and 109–114 (in magenta) are shown. (C) Lysates of NCI‐H1299 cells transfected with the indicated plasmids were immunoprecipitated with anti‐FLAG antibody and analyzed by western blot using the indicated antibodies (representative of n = 3 independent experiments).

Article Snippet: Briefly, cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with mouse anti‐p53 (Santa Cruz Biotechnology, #sc‐126) and rabbit anti‐FLAG (Proteintech, #80010‐1‐RR; probing for FLAG‐tagged PDPK1) primary antibodies.

Techniques: Mutagenesis, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Biomarker Discovery

Journal: Autophagy

Article Title: CHCHD2 and CHCHD10 promoted autophagic clearance of protein aggregates via GABARAPs

doi: 10.1080/15548627.2026.2678427

Figure Lengend Snippet: Antibodies.

Article Snippet: FLAG , Proteintech Group , 80010-1-rr.

Techniques: Cell-Signaling, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Genomic distribution of SP9 ChIP-seq peaks based on data from ( ; ). Peaks localize predominantly to enhancer-like regions (77%), with smaller fractions at promoters (6.2%) and other regions (16.8%). B , Motif enrichment within SP9 peaks. Promoter-associated peaks (left) are enriched for GC-rich motifs recognized by KLF and SP factors, whereas enhancer-like peaks (right) are enriched for TAATT motifs recognized by DLX homeobox factors. Within each block: motif logos (left), − log 10 𝑃 -value and target/background percentage of peaks containing the motif (middle), and log 2 enrichment (right). C , Schematic of the motif scan-based classification of SP9 peaks. Peaks were grouped based on the presence of an SP motif only (direct binding), a DLX motif only (indirect binding via DLX), or both (cobinding). D , Coarse genomic annotation (enhancer, transcription start site (TSS), or other) of the four SP9 peak categories defined in (C): SP9+DLX (both motifs present), DLX (DLX motif only), Other, and SP9 (SP motif only). E , Fine genomic annotation (exon, intron, intergenic, promoter, UTR, other) of the same four peak categories. F , Intersection of DLX2 and SP9 ChIP-seq peaks from E13.5 mouse GE, based on data from ( ; ; ). Bar plot shows the number of overlapping and non-overlapping peaks per category (DLX2 only, overlap, SP9 only). G , Whole-mount X-Gal staining (blue) of E11.5 transgenic mouse embryos carrying VISTA enhancer reporters hs119 (near Arx ), hs170 (near Fign ), hs883 (near Sox6 ), and hs298 (near Dlx6os1 ) used in the luciferase assays in panels K-N and shown as genome browser tracks in panel H. Images from . H , Genome browser tracks showing ChIP-seq signal for SP9, DLX2, and DLX5 in E13.5 mouse GE at the Sp9 and Six3 promoters (highlighted in grey) and the VISTA enhancers hs119, hs170, hs883, and hs298 (highlighted in blue). Numbers below each track indicate the count of SP and DLX motifs within the selected regulatory element. Data from ( ; ; ). I-N , Luciferase reporter activity in N2A cells transfected with combinations of Sp9 and Dlx2 , Dlx5 , or Dlx6 as indicated, driven by the Sp9 promoter ( I ), Six3 promoter ( J ), or enhancer regions hs119 of Arx ( K ), hs170 of Fign ( L ), hs883 of Sox6 ( M ), and hs298 of Dlx6os1 ( N ). Bars represent mean ± s.e.m. of 9 (I,J,L-N) or 12 (K) replicates from 3 (I,J,L-N) or 4 (K) independent batches performed in triplicate; points indicate batch means. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6.

Article Snippet: Trypsin was neutralized with 1 % BSA in PBS, and cells were collected by centrifugation at 400 x g for 5 min. For each sample, 500,000 cells were processed and incubated with the following antibodies: Anti-V5 (SP9) (Thermo Fisher Scientific, R96025), Anti-FLAG (DLX5) (EpiCypher, 13-2031), Anti-H3K4me3 (EpiCypher, 13-0041), and AntiIgG (EpiCypher, 13-0042) as a negative control.

Techniques: ChIP-sequencing, Blocking Assay, Binding Assay, Staining, Transgenic Assay, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Schematic of C-terminally V5-tagged SP9 constructs: wild-type (WT) SP9, an N-terminal deletion mutant (NΔSP9), and a zinc finger (ZF) domain deletion mutant (SP9ΔZF). The nine-amino-acid transactivation domain (9aaTAD), the three ZF domains, and the position of the patient-derived SP9*378 variant are indicated. B , Co-immunoprecipitation (CoIP) from nuclear lysates of HEK293FT cells co-transfected with DLX5-FLAG and either SP9-V5 WT, SP9*378-V5, or NΔSP9V5. Left: input fractions probed with anti-SP9 (top) and anti-DLX5 (bottom). Right: CoIP using anti-FLAG beads, probed with anti-SP9 (top) and anti-DLX5 (bottom). C , CoIP from whole-cell lysates of HEK293FT cells co-transfected with DLX5-FLAG and either SP9-V5 WT or SP9ΔZF-V5. Left: input fractions probed with anti-V5 (top) and anti-DLX5 (bottom). Right: CoIP using anti-FLAG beads, probed with anti-V5 (the anti-SP9 epitope lies within the deleted ZF region, requiring V5 detection; top) and anti-DLX5 (bottom). D , Luciferase reporter activity driven by the hs298 enhancer of Dlx6os1 in N2A cells co-transfected with Dlx5 and either Sp9 , Sp9*378 , N Δ Sp9 , or Sp9 Δ ZF . Bars represent mean ± s.e.m. of9replicatesfrom3independentbatchesperformedintriplicate; pointsindicatebatchmeans. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6. E , Label-free quantitative mass spectrometry (MS) of proteins captured by DNA pull-down using a 46-bp fragment of the hs298 enhancer of Dlx6os1 versus the same fragment with the TAATT motifs scrambled, from E14.5 ganglionic eminence (GE) nuclear lysates. Proteins with p ≤ 0.05 and log 2 LFQ FC ≥ 1.5 were considered significantly enriched (n = 25). Transcription factors (TFs) are indicated in black. F , Volcano plot of proteins enriched in anti-SP9 CoIP-MS from E14.5 GE nuclear lysates, relative to IgG controls. Proteins with log 2 FC ≥ 1.5 and p ≤ 0.05 were considered significantly enriched (n = 223). Components of HDAC1/2-containing complexes are highlighted in blue; TFs are indicated in black. 𝑃 -values were calculated using a two-sided Student’s t-test with permutation-based false discovery rate (FDR) correction. G , Gene Ontology (GO) molecular function enrichment analysis of proteins identified by anti-SP9 CoIP-MS. H , Heatmap of ChIP-seq signal intensity across SP9-bound regions for SP9 ( ; ), DLX2, histone modifications (H3K27ac, H3K4me1, H3K4me3) , GTF2I , and NuRD complex subunits (MBD3, CHD4, RBBP4, RBBP7, HDAC1, HDAC2) . Signal is plotted over a ±5 kb window centered on SP9 peak summits and organized by k-means clustering (5 clusters), separating SP9-bound regions by their co-binding patterns with DLX2, NuRD subunits, and active histone marks. All datasets are from mouse E13.5 GE except GTF2I, which is from E13.5 whole brain.

Article Snippet: Trypsin was neutralized with 1 % BSA in PBS, and cells were collected by centrifugation at 400 x g for 5 min. For each sample, 500,000 cells were processed and incubated with the following antibodies: Anti-V5 (SP9) (Thermo Fisher Scientific, R96025), Anti-FLAG (DLX5) (EpiCypher, 13-2031), Anti-H3K4me3 (EpiCypher, 13-0041), and AntiIgG (EpiCypher, 13-0042) as a negative control.

Techniques: Construct, Mutagenesis, Derivative Assay, Variant Assay, Immunoprecipitation, Transfection, Luciferase, Activity Assay, Mass Spectrometry, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Schematic of the transcription factor (TF) overexpression strategy in N2A cells. Cells were transfected with SP9 alone or co-expressed with WT DLX5 (overexpression experiment 1, OE1) or with the N-terminal deletion mutant NΔDLX5 (OE2), followed by CUT&RUN profiling. B , Domain organization of SP9, DLX5, and NΔDLX5, highlighting the zinc finger (ZF) and homeobox domains. C , Heatmaps of SP9 CUT&RUN signal (anti-V5) centered on SP9 peak regions (±5 kb), clustered into four groups (C1-C4) by k-means. Conditions: SP9 alone, SP9+DLX5 (OE1), SP9+NΔDLX5 (OE2). D , Aggregate signal plots for each cluster (C1-C4) showing normalized SP9 binding profiles at peak centers under the three conditions in (C). E , Top enriched motifs identified by motif analysis of peaks in each cluster, with associated 𝑃 -values. F , Genomic annotation of SP9-bound peaks across clusters. Left: distribution across promoter, exon, intron, intergenic, and other regions. Right: distance to transcription start sites (TSS). G , Genome browser tracks at three representative loci showing SP9 binding under OE1 and OE2 conditions, illustrating loss of SP9 occupancy upon DLX5 coexpression at SP-motif-containing sites and rescue with NΔDLX5, as well as gain of SP9 occupancy at DLX-motif-containing sites with WT DLX5. H , Luciferase reporter activity driven by the Six3 promoter in N2A cells transfected with combinations of Sp9 , Dlx5 , and N Δ Dlx5 as indicated. I , Volcano plot of co-immunoprecipitation followed by mass spectrometry (CoIP-MS) using anti-V5 in N2A cells co-transfected with Sp9-V5 and Dlx5-FLAG , relative to IgG controls. Proteins with log 2 FC > 1.5 and p < 0.05 were considered significantly enriched (n = 2254). NuRD complex components are highlighted in blue, transcription factors (TFs) in black, and AP-1 family TFs in green. J , Volcano plot comparing the anti-V5 CoIP-MS interactomes between SP9+DLX5 and SP9+NΔDLX5 conditions in N2A cells, using the same cutoffs as in (I). I,J , 𝑃 -values were calculated using a two-sided Student’s t-test with permutation-based false discovery rate (FDR) correction. K , Luciferase reporter activity driven by the Six3 promoter in N2A cells transfected with varying amounts (ng plasmid DNA) of SP9 (S) and DLX5 (D), showing dose-dependent transcriptional activation. H-K , Bars represent mean ± s.e.m. of 9 replicates from 3 independent batches performed in triplicate; points indicate batch means. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6. L , Proposed mechanism. When SP9 levels exceed DLX (SP9≫DLX), SP9 binds directly at GC-rich SP motifs. When SP9 and DLX are comparable or DLX is in excess (SP9≈DLX or SP9 < DLX), DLX5 sequesters SP9 to TAATT-containing DLX motifs through the DLX5 N-terminal domain, away from its direct SP-motif binding sites. NΔDLX5, which cannot interact with SP9, fails to redirect SP9 to DLX motifs, restoring SP9 binding at SP motifs.

Article Snippet: Trypsin was neutralized with 1 % BSA in PBS, and cells were collected by centrifugation at 400 x g for 5 min. For each sample, 500,000 cells were processed and incubated with the following antibodies: Anti-V5 (SP9) (Thermo Fisher Scientific, R96025), Anti-FLAG (DLX5) (EpiCypher, 13-2031), Anti-H3K4me3 (EpiCypher, 13-0041), and AntiIgG (EpiCypher, 13-0042) as a negative control.

Techniques: Over Expression, Transfection, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Mass Spectrometry, Plasmid Preparation, Activation Assay

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , UMAP visualization of the GABAergic single-cell transcriptomic dataset, colored by the per-cell ratio of normalized Sp9 expression counts to the summed expression of Dlx1, Dlx2, Dlx5 , and Dlx6 (Ratio Sp9 :Σ Dlx ). A subset of cells in the shared trunk before the D1-D2 MSN bifurcation already shows elevated ratios, possibly representing cells biased toward the D2 MSN fate. B , Expression dynamics along the projection neuron pseudotime trajectory for Sp9 (left), summed Dlx1/2/5/6 expression (middle), and their ratio Sp9 :Σ Dlx (right). Lines indicate the D1 MSN trajectory (blue), the D2 MSN trajectory (orange), and the shared trajectory of progenitors and early postmitotic precursors (green). Shaded region marks the postmitotic branch following the D1-D2 MSN bifurcation. C , Proposed model of stoichiometric SP9-DLX factors control of MSN fate. In progenitors and early postmitotic precursors (SP9≤DLX),SP9-DLXfactorcomplexesoccupyDLX-motif-containingdistalregulatoryelements(REs) ofD1andD2MSNfategenes; GC-richpromotersofD2MSNgenessuchas Six3 remainunbound. Along the D2 MSN trajectory (SP9 > DLX), the relative increase in SP9 enables two simultaneous effects: direct binding of SP9 at GC-rich Six3 promoter activates D2 MSN-fate gene expression, while SP9-DLX factor complexes at distal D1 MSN-gene REs recruit the NuRD complex (CHD, MBD, RBBP4/7, HDAC1/2) to repress D1 MSN-fate genes. Along the D1 MSN trajectory (SP9 << DLX), SP9 is absent from these REs; DLX factors remains bound at distal TAATT sites, and D1 MSN-fate genes such as Arx are activated through other transcription factors (TFs) at their proximal REs.

Article Snippet: Trypsin was neutralized with 1 % BSA in PBS, and cells were collected by centrifugation at 400 x g for 5 min. For each sample, 500,000 cells were processed and incubated with the following antibodies: Anti-V5 (SP9) (Thermo Fisher Scientific, R96025), Anti-FLAG (DLX5) (EpiCypher, 13-2031), Anti-H3K4me3 (EpiCypher, 13-0041), and AntiIgG (EpiCypher, 13-0042) as a negative control.

Techniques: Single Cell, Expressing, Control, Binding Assay, Gene Expression