rabbit anti sp1 antibody  (Proteintech)

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: Repeated NTG administration induces migraine-like behaviors and elevates SP1, TET1, and KIF1A expression in the SP5C. ( A ) Experimental timelines for NTG-induced migraine modeling, SP1 inhibition (MTM), and Kif1a manipulation (Ad- Kif1a or Ad-mCherry). Behavioral testing was performed as indicated, followed by tissue collection on day 10. ( B – D ) Repeated NTG administration induced sustained nociceptive hypersensitivity, reflected by reduced mechanical allodynia (HML and HMT) and thermal hyperalgesia (PMT) ( n = 8 mice per group). ( E ) Representative WB images showing increased protein levels of TET1, KIF1A, SP1, c-Fos, and CGRP in the SP5C after NTG treatment. ( F ) Quantification of TET1, KIF1A, SP1, c-Fos, and CGRP protein levels normalized to β-actin ( n = 4 mice per group). ( G ) RT-qPCR analysis of Kif1a mRNA expression after NTG treatment the SP5C. ( n = 4mice per group). ( H ) Representative images of IF staining images showing CGRP expression in the SP5C. Nuclei were counterstained with DAPI (blue). Scale bars: 200 μm. ( I ) Quantification of the fluorescence intensity showing the significant upregulation of CGRP in NTG-treated mice ( n = 4 mice per group). Statistical analysis: Two-way ANOVA for ( B - D ); unpaired two-tailed t test for ( F , G , I ). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the VEH group. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Staining, Fluorescence, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: Pharmacological inhibition of SP1 suppresses KIF1A signaling and CGRP expression. ( A ) Representative WB images showing time-dependent suppression of SP1 protein expression following MTM treatment (2, 6, 12, and 24 h) compared with control cells. ( B ) Quantification of SP1 protein levels normalized to β-actin ( n = 4). ( C ) Representative WB images showing reduced protein expression of KIF1A, SP1, c-Fos, and CGRP after MTM treatment compared to DMSO controls. ( D ) Quantitative analysis of CGRP, c-Fos, SP1, and KIF1A protein levels following MTM treatment ( n = 4). ( E ) RT-qPCR analysis showing decreased Kif1a mRNA expression in the MTM-treated group relative to controls ( n = 4). ( F ) Representative IF images showing reduced CGRP expression (green) after MTM treatment. Nuclei were counterstained with DAPI (blue). Scale bar = 100 μm. Statistical significance: One-way ANOVA for ( B ); unpaired two-tailed t-test for ( D , E ). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DMSO group. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Inhibition, Expressing, Control, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: SP1 regulates KIF1A expression through TET1-dependent epigenetic mechanisms. ( A ) Representative WB showing reduced TET1 protein expression following MTM treatment compared to DMSO. ( B ) Quantification of TET1 protein levels normalized to β-actin ( n = 4). ( C ) Screening of Tet1 -targeting sequences in N2a cells. ( D ) Quantification of TET1 protein levels after Tet1 siRNA transfection ( n = 4) showed that sequence 2 produced the strongest knockdown. ( E ) RT-qPCR analysis showing sequence 2 most effectively decreased Tet1 mRNA ( n = 4). (F ) Representative WB showing reduced KIF1A, SP1, c-Fos, and CGRP after Tet1 siRNA transfection. ( G ) Quantification of CGRP, c-Fos, SP1, and KIF1A protein levels following Tet1 siRNA transfection ( n = 4). ( H ) RT-qPCR showing decreased Kif1a mRNA expression in Tet1 siRNA–treated cells ( n = 4). ( I ) IF images showing reduced CGRP expression (green) after Tet1 siRNA transfection. Scale bar = 100 μm. ( J ) Co-IP showing SP1 and TET1 interaction after immunoprecipitation with anti-SP1 antibody. ( K ) Co-IP showing SP1 and TET1 interaction after immunoprecipitation with anti-TET1 antibody. ( L ) Dual-luciferase reporter assay showing increased Kif1a promoter–driven luciferase activity after SP1 overexpression ( n = 3). ( M ) ChIP-qPCR showing SP1 and TET1 enrichment at the Kif1a promoter, reduced after sequential Re-ChIP with TET1 antibodies ( n = 3). Statistical significance: Unpaired two-tailed t-test for ( A , G , H ); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO or MS siRNA. One-way ANOVA for ( D , E ); Two-way ANOVA for ( L ); * p < 0.05, ** p < 0.01, ** p < 0.001 compared the corresponding control groups, as indicated. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, Transfection, Sequencing, Produced, Knockdown, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Over Expression, ChIP-qPCR, Two Tailed Test, Control

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: Effects of MTM treatment on TET1, KIF1A, SP1, and CGRP expression in the NTG-induced migraine model. ( A – C ) NTG induced mechanical allodynia (decreased HML and HMT) and thermal hyperalgesia (decreased PMT). MTM treatment significantly attenuated NTG-induced nociception compared with NTG + DMSO ( n = 8 mice per group). ( D ) Representative WB images showing protein expression levels of TET1, KIF1A, SP1, c-Fos, and CGRP in the VEH and NTG groups treated with DMSO or MTM. ( E ) Quantification of TET1, KIF1A, SP1, c-Fos, and CGRP protein levels normalized to β-actin ( n = 4 mice per group). ( F ) Quantification of Kif1a mRNA expression in the VEH and NTG groups treated with DMSO or MTM ( n = 4 mice per group). ( G ) Representative IF images showing CGRP expression (green) in the VEH and NTG groups treated with DMSO or MTM. Nuclei were counterstained with DAPI (blue). Scale bar = 200 μm. ( H ) Quantification of CGRP fluorescence intensity across groups ( n = 4 mice per group). ( I ) Methylation analysis of the Kif1a promoter in the VEH and NTG groups treated with DMSO or MTM, showing alterations in DNA methylation patterns. (L) Quantitative analysis of Kif1a promoter DNA methylation ( n = 3 mice per group). Statistical significance: Two-way ANOVA for ( A – C ); One-way ANOVA for ( E , F , H , J ); * p < 0.05, ** p < 0.01, *** p < 0.001 compared the VEH+DMSO groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the NTG+DMSO groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, Fluorescence, Methylation, DNA Methylation Assay

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: SP1 inhibition suppresses synaptic protein expression in vitro and in vivo. ( A ) Representative WB images showing reduced expression of the synaptic proteins PSD-95, Syt1, and SYP following MTM treatment compared with DMSO in N2a cells. ( B ) Quantification of PSD-95, Syt1, and SYP protein levels normalized to β-actin ( n = 4). ( C – E ) Representative IF images showing decreased expression of SYP ( C ), Syt1 ( D ), and PSD-95 ( E ) (green) after MTM treatment. Nuclei were counterstained with DAPI (blue). ( F ) Representative WB images showing PSD-95, Syt1, and SYP protein expression in the SP5C of VEH and NTG mice treated with DMSO or MTM. ( G ) Quantification of PSD-95, Syt1, and SYP protein levels normalized to β-actin ( n = 4 mice per group). ( H ) Representative IF images showing Syt1 expression (green) in the SP5C of VEH and NTG mice treated with DMSO or MTM. Scale bar = 100 μm. ( I ) Quantification of Syt1 fluorescence intensity across groups ( n = 4 mice per group). ( J ) Representative IF images showing PSD-95 expression (green) in the SP5C of VEH and NTG mice treated with DMSO or MTM. Scale bar = 100 μm. ( K ) Quantification of PSD-95 fluorescence intensity across groups ( n = 4 mice per group). Statistical significance: Unpaired two-tailed t-test for (B); * p < 0.05, ** p < 0.01, ** p < 0.001 vs. DMSO; One-way ANOVA for ( G , I , K ), * p < 0.05, ** p < 0.01, ** p < 0.001 compared the VEH+DMSO groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the NTG+DMSO groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Inhibition, Expressing, In Vitro, In Vivo, Fluorescence, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: TET1 mediates SP1-dependent regulation of synaptic protein expression and CGRP signaling in vitro. ( A ) Representative WB images showing reduced expression of the synaptic proteins PSD-95, Syt1, and SYP following Tet1 siRNA transfection compared with MS siRNA controls. ( B ) Quantification of PSD-95, Syt1, and SYP protein levels normalized to β-actin ( n = 4). ( C – E ) Representative IF images showing decreased expression of SYP ( C ), Syt1 ( D ), and PSD-95 ( E ) (green) after Tet1 siRNA transfection. ( F ) Representative WB images showing expression of KIF1A, PSD-95, Syt1, SYP, and CGRP in cells transfected with pcDNA3.1- Tet1 or control plasmid, with or without MTM treatment. ( G ) Quantification of CGRP, SYP, Syt1, PSD-95, and KIF1A protein levels normalized to β-actin ( n = 4). ( H ) RT-qPCR analysis showing Kif1a mRNA expression following pcDNA3.1- Tet1 transfection in the presence or absence of MTM ( n = 4). ( I ) Representative IF images showing CGRP expression (green) under the indicated conditions. Scale bar = 100 μm. Statistical significance: Unpaired two-tailed t-test for ( B ); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO; One-way ANOVA for (G, H), * p < 0.05, ** p < 0.01, ** p < 0.001 compared the control groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the MTM groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, In Vitro, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: KIF1A mediates SP1 inhibition–induced synaptic ultrastructural and dendritic spine remodeling in the SP5C. ( A ) Representative TEM images of synaptic ultrastructure in the SP5C from the VEH, NTG, NTG + MTM, NTG + MTM + Ad- Kif1a , and NTG + MTM + Ad-mCherry groups. Dashed boxes indicate synaptic regions shown at higher magnification (Zoom). Scale bars = 500 nm. ( B ) Quantification of synaptic cleft width. NTG reduced synaptic cleft width, which was reversed by MTM and restored by Ad- Kif1a . ( C ) Quantification of synaptic interface curvature. NTG increased synaptic interface curvature; this increase was attenuated by MTM and restored by Ad- Kif1a . ( D ) Quantification of PSD thickness. NTG increased PSD thickness, which was reduced by MTM and re-elevated by Ad- Kif1a but not by Ad-mCherry. ( E ) Representative Golgi-Cox–stained images of dendritic morphology and spines in SP5C neurons. Enlarged images (Zoom) show dendritic segments used for spine analysis. Scale bars = 50 μm. ( F ) Quantification of dendritic spine density, expressed as the number of spines per 20 μm of dendritic length. NTG increased spine density, which was partially reversed by MTM and restored by Ad- Kif1a . Statistical significance: One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 compared the VEH groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the NTG groups; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 compared the NTG+MTM groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Inhibition, Staining

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: AR transactivation is linked to increased chromatin accessibility and SP1 footprint dynamics. Three‐week‐old female C57BL/6J mice were subcutaneously implanted with silicone tubes (1 cm) containing dehydroepiandrosterone (DHEA) for 35 days to establish a PCOS model, the equal‐length empty silicone tubes served as the control (Ctrl). Ovarian tissues were collected for ATAC‐seq and RNA‐seq analysis (n = 3 in each group). (a) Heatmap of normalized expression (FPKM) for 15 key genes associated with estrogen signaling, cortisol synthesis and secretion, HIF‐1α signaling, and oocyte meiosis in Ctrl and DHEA‐treated mice by RNA‐seq analysis. (b) Open chromatin regions surrounding gene transcription start sites (TSSs) in ovarian tissues from Ctrl and DHEA‐treated mice by ATAC‐seq analysis. (c) Pie chart depicting the peaks annotation identified in the ATAC‐seq analysis. The chart illustrates the distribution of accessible chromatin regions across various functional elements in the genome. (d) Venn diagram illustrating the overlap between gene with increased peaks (ATAC‐seq, p < 0.05 ) and genes with increased expression (RNA‐seq, |log2FC| > 1, p < 0.05 ) in Ctrl vs. DHEA‐treated mice. (e) Peak plot showing the ATAC‐seq peaks (normalized reads) and RNA‐seq mRNA expression (FPKM) at the Ar locus (ChrX: 98147619–98152019) in Ctrl (blue) vs. DHEA‐treated (red) mice. The peaks exhibiting a specific increase upon DHEA treatment are indicated by gray boxes and red asterisk. (f) A heatmap displays the top six transcription factors (TFs) binding to the Ar promoter region in the ATAC‐seq analysis, along with their mRNA expression identified by RNA‐seq analysis, and the predicted TF motifs were shown on the right. (g) Aggregated footprint plot of SP1. The aggregated plot depicts the predicted SP1 binding motifs across 852 total possibilities, comparing the Ctrl and DHEA conditions. The dashed lines indicate the boundaries of the SP1 motif at the Ar promoter. The plot visualizes the change in chromatin accessibility and transcription factor (TF) footprints across the different treatments. (h) Western blotting (the left side) and ChIP assay (the right side). Western blotting assay, the ovarian tissue homogenates from Ctrl and DHEA‐treated mice were assayed for SP1, p300, BRD4 and GAPDH. ChIP assay, the ovarian tissue homogenates were immunoprecipitated with isoform‐matched immunoglobulin or antibodies to SP1, p300 or BRD4, respectively, and then the genomic DNA (Input) and the antibody‐bound DNAs were PCR‐amplified with primers covering the SP1 motif on Ar promoter. The PCR products were analyzed on 1.5% agarose gels. (i) Western blotting. KGN cells were treated with DHEA (25 µ m , 48 h) with or without Plicamycin (Plica, 100 n m , 24 h), and then cell lysates were assayed for SP1, AR and GAPDH. The quantification graph is shown below. Data were presented as means ± SEM. n = 4, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Antibodies against SP1 (A19649, ABclonal), p300 (ab275378, Abcam), BRD4 (83375S, CST), H3K18ac (13998T, CST), and H3K27ac (8173T, CST) were used for immunoprecipitation.

Techniques: Control, RNA Sequencing, Expressing, Functional Assay, Binding Assay, Western Blot, Immunoprecipitation, Amplification

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: p300 forms a transcriptional complexes with SP1 and BRD4 to drive AR activation. (a) Scatter plots showing EP300 ‐ AR ( Ep300 ‐ Ar ) and SP1 ‐ AR ( Sp1 ‐ Ar ) expression correlations in human ovarian granulosa cells (the left panel) and murine ovaries (the right panel). (b) ChIP assay. KGN cells were treated with DHEA (25 µ m , 48 h) in presence or absence of C646 (10 µ m , 24 h) or Plicamycin (Pli, 100 n m , 24 h), respectively, and then cell lysates were immunoprecipitated with isoform‐matched immunoglobulin or antibodies to p300, SP1, H3K18ac, or H3K27ac, respectively, and then the genomic DNA (Input) and the antibody‐bound DNAs were PCR‐amplified with primers covering the SP1 motif on AR promoter. The PCR products were analyzed on 1.5% agarose gels. (c) Quantification of (b). Data were presented as Box‐and‐whisker plots with data points and as means ± SEM; n = 4, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (d) Co‐IP assay. KGN cells were treated with DHEA (25 µ m , 48 h) with or without C646 (10 µ m , 24 h), and then cell lysates were immunoprecipitated with isoform‐matched immunoglobulin (Ig) or antibody (IP Ab) to p300, BRD4, or SP1, and then immunoprecipitants were assessed for p300, BRD4, or SP1 by western blotting reciprocally (the upper panel). The non‐IP lysates were assayed for p300, BRD4, SP1, and GAPDH as input controls. (e) Quantification of the Input bands in (d). Data were presented as means ± SEM; n = 3, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (f) Quantification of the IP bands in (d). Data were presented as means ± SEM; n = 3, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (g) KGN cells were treated with DHEA (25 µ m , 48 h) with or without C646 (10 µ m , 24 h), and then were stained for p300 (red), BRD4 (green) and SP1 (magenta) by multiplex immunofluorescence (mIF) staining. The colocalization analysis peak plot is shown below. (h) HEK293T cells were transfected with an AR promoter‐luciferase reporter AR‐luc (GGGGAGGGG) or a mutant AR promoter reporter ARmut‐luc (TAATGTAAT) plus a renilla luciferase reporter for 24 h, in presence or absence of a plasmid overexpressing SP1 (Gv‐SP1). At the same time, cells were treated with or without C646 (10 µ m , 24 h) or Plicamycin (Plica, 100 n m , 24 h), respectively. Then cell lysates were assayed for SP1 and GAPDH (Western blotting) or luciferase activities. The relative luciferase activities of fold changes were presented. Data were presented as means ± SEM; n = 4, *P < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Antibodies against SP1 (A19649, ABclonal), p300 (ab275378, Abcam), BRD4 (83375S, CST), H3K18ac (13998T, CST), and H3K27ac (8173T, CST) were used for immunoprecipitation.

Techniques: Activation Assay, Expressing, Immunoprecipitation, Amplification, Whisker Assay, Co-Immunoprecipitation Assay, Western Blot, Staining, Multiplex Assay, Immunofluorescence, Transfection, Luciferase, Mutagenesis, Plasmid Preparation

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: AR activation undermines the anti‐fibrosis effects of p300 inhibition in ovaries of PCOS ovaries. Three‐week‐old female C57BL/6J mice were accepted high‐fat diet (HF, 60% fat) for 3 months, and mice receiving Control vehicle (Ctrl) or DHT (1.66 mg/kg, 10 days) were grouped into Control (Ctrl), C646, high‐fat diet (HF), and C646‐treated HF mice (ten mice in each group). (a) Representative microphotographs of ovarian sections stained by Hematoxylin‐eosin (the left panel) and Masson trichrome (the right panel). The corpora lutea and collagen deposition were indicated by asterisk or arrows, respectively. (b) Quantitation of corpora lutea ratio and fibrosis staining of (a). Data were presented as Box‐and‐whisker plots with data points. Data were presented as means ± SEM; n = 6, *p < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test. (c) Western blotting. The ovarian tissues from the experimental mice in (a) were assayed for p300, AR, Collagen I (Col1α), α‐SMA, and GAPDH. Two randomly‐selected samples from each group were shown. (d) Quantification of (c). Data were presented as means ± SEM. n = 6, *p < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test. (e) A schematic of sequential p300 induction of histone 3 acetylation, formation of p300/BRD4/SP1 complex and AR transactivation, leading to PCOS. P300 inhibition with A‐485/C646 blocks the process.

Article Snippet: Antibodies against SP1 (A19649, ABclonal), p300 (ab275378, Abcam), BRD4 (83375S, CST), H3K18ac (13998T, CST), and H3K27ac (8173T, CST) were used for immunoprecipitation.

Techniques: Activation Assay, Inhibition, Control, Staining, Quantitation Assay, Whisker Assay, Western Blot