antibodies against smad4  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

doi: 10.64898/2026.03.28.714793

Figure Lengend Snippet: (A) The proportion of samples with mutations in TP53 , SMAD4 , KRAS , and STK11 in TRACERX LUSC cohort was shown by bar plots. (B) SMAD4 mRNA expression (log2 scale) between different genotypes of LUSC patients. (C) Survival curves showed the relationship between SMAD4 expression levels and prognosis. (D) Western Blot analysis of SMAD4 protein levels in Smad4 knockout (gRNA- Smad4 ) and non-knockout (gRNA-Control) in MSCC LP.3 cell lines. GAPDH served as the loading control. (E) Colony formation assay under Smad4 knockout or non-knockout conditions and the quantification of the relative gray value ratio in MSCC LP.3 cell line using ImageJ. Data were presented as mean ± SD. (F) Tumor size and weight of MSCC LP.3 gRNA-Control tumor groups and gRNA- Smad4 tumor groups in vivo. (G) Phenotype analysis by H&E staining and IHC staining of LUSC markers KRT5 and TP63, LUAD marker TTF1, and cell proliferation marker Ki67 in xenograft tumors with or without Smad4 knockout. The scale bar presented 20µm. (H) Mouse model design workflow and phenotype statistics of LUSC in different genetic backgrounds. (I) LUSC markers KRT5 and TP63, LUAD marker TTF1, and SMAD4 IHC staining along with H&E staining in CCSP +/+ Lkb1 f/f Smad4 f/f , CCSP iCre Smad4 f/f , CCSP iCre Lkb1 f/f , and CCSP iCre Lkb1 f/f Smad4 f/f mice. (J) IHC staining of SMAD4, LUSC markers KRT5 and TP63, and LUAD marker TTF1, and H&E staining in Adeno-Cre KRAS G12D Trp53 f/f and Adeno-Cre Smad4 f/f KRAS G12D Trp53 f/f mice.

Article Snippet: After blocking with 5% non-fat milk for 1 hour at room temperature (RT), membranes were incubated overnight at 4 °C with primary antibodies against SMAD4 (1:1000, CST#38454), SOX2 (1:250, CST#14962), EP300 (1:500, Sigma-Merk 05-257), GAPDH (1:1000, CST#2118), or β-Actin (1:500, HANGZHOU FUDE BIOLOGICAL TECHNOLOGY CO., LTD., FD0060), followed by HRP-conjugated secondary antibodies (1:5000, Easy bio, BE0101 and BE0102).

Techniques: Expressing, Western Blot, Knock-Out, Control, Colony Assay, In Vivo, Staining, Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

doi: 10.64898/2026.03.28.714793

Figure Lengend Snippet: (A) Differentially expressed genes between MSCC LP.3 gRNA- Smad4 and gRNA-Control illustrated by the volcano plot. (B) The top enriched signaling pathways obtained from differentially expressed genes by GO enrichment. (C) Pathways enriched from differentially expressed genes by GSEA enrichment. (D) Gene Interaction Network in the Cell Cycle Process Extracted from the STRING Database. (E) Western Blot analysis of SOX 2 protein levels in Smad4 knockout (gRNA- Smad4 ) and non-knockout (gRNA-Control) in MSCC LP.3 cell lines. β-actin served as a loading control. (F) RT-qPCR analysis of Sox2 mRNA in Smad4 knockout (gRNA-Smad4) and non-knockout (gRNA-Control) in MSCC LP.3 cell lines. 18S served as the RT-qPCR loading control. (G) Smad4 expression levels and Sox2 expression levels at different time points following Smad4 siRNA knockdown in MSCC LP.3 cell lines. (H) Sox2 expression level before and after overexpression of Smad4 ( Smad4_ OV). (I) IHC staining of SOX2 in xenograft tumors with or without Smad4 knockout and mouse models with different genetic backgrounds.

Article Snippet: After blocking with 5% non-fat milk for 1 hour at room temperature (RT), membranes were incubated overnight at 4 °C with primary antibodies against SMAD4 (1:1000, CST#38454), SOX2 (1:250, CST#14962), EP300 (1:500, Sigma-Merk 05-257), GAPDH (1:1000, CST#2118), or β-Actin (1:500, HANGZHOU FUDE BIOLOGICAL TECHNOLOGY CO., LTD., FD0060), followed by HRP-conjugated secondary antibodies (1:5000, Easy bio, BE0101 and BE0102).

Techniques: Control, Protein-Protein interactions, Western Blot, Knock-Out, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Immunohistochemistry

Journal: bioRxiv

Article Title: A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

doi: 10.64898/2026.03.28.714793

Figure Lengend Snippet: (A) Profiling of SMAD4 and H3K27ac binding on DEGs in MSCC LP.3 . (B) The location distribution of SMAD4 binding on different genomic regions. (C) Statistics of SMAD4 binding distribution around gene transcription start sites (TSS). (D) Venn plot of genes regulated by SMAD4 binding and H3K27ac modification. (E) Liftover H3K27ac ChIP-Seq peak regions of normal human lung tissue from the reference genome hg19 to the mouse reference genome mm9, matching with the H3K27ac CUT&RUN result in MSCC LP.3 shown in IGV browser. (F) RT-qPCR of Sox2 expression followed by the knockout of the enhancer region of Sox2 -associated loop in MSCC LP.3 gRNA- Smad4 cell line. (G) Colony formation assay after the knockout enhancer region of Sox2 -associated loops in MSCC LP.3 gRNA- Smad4 cell line and the quantification of the relative gray value ratio in MSCC LP.3 cell line using ImageJ. (H) Sox2 expression before and after Sox2 -associated enhancer region targeted by antisense oligonucleotides (ASO) in MSCC LP.3 with or without Smad4 KO. Cells were incubated with 15nM ASO oligos for 48□h. (I) The 3C-qPCR analysis of the frequency of Sox2 -associated loop by 3C-qPCR after Smad4 knockout. (J) The 3C-qPCR analysis of the frequency of Sox2 -associated loop by 3C-qPCR after Smad4 overexpression ( Smad4 _OV). (K) Hi-C analysis of BEAS-2B gRNA- PTEN cell line with or without SMAD4 knockout. (L) Hi-C heatmap around the SOX2 locus exhibiting the location of the SOX2 -associated loops in BEAS-2B gRNA- PTEN cells with or without SMAD4 knockout.

Article Snippet: After blocking with 5% non-fat milk for 1 hour at room temperature (RT), membranes were incubated overnight at 4 °C with primary antibodies against SMAD4 (1:1000, CST#38454), SOX2 (1:250, CST#14962), EP300 (1:500, Sigma-Merk 05-257), GAPDH (1:1000, CST#2118), or β-Actin (1:500, HANGZHOU FUDE BIOLOGICAL TECHNOLOGY CO., LTD., FD0060), followed by HRP-conjugated secondary antibodies (1:5000, Easy bio, BE0101 and BE0102).

Techniques: Binding Assay, Modification, ChIP-sequencing, Quantitative RT-PCR, Expressing, Knock-Out, Colony Assay, Incubation, Over Expression, Hi-C

Journal: bioRxiv

Article Title: A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

doi: 10.64898/2026.03.28.714793

Figure Lengend Snippet: (A) Overlap analysis of proteins that interact with the SMAD4 protein and the TFs whose motifs were enriched at Sox2 -associated loop target regions. The SMAD4 interactors were identified from the BioGRID database. The overlapped proteins were ranked by the evidence of interaction with SMAD4 according to BioGRID. (B) The ChIP-Seq peaks of the top three candidates enriched in Sox2 -associated loop regions. Anchor regions of the Sox2 -associated loop were shown in green background, and the EP300 binding site were indicated by red arrows. The ChIP-Seq data were from the ChIP-atlas database, and all collected cell types of the candidate proteins were selected. (C) Co-IP confirmed the interaction between EP300 and SMAD4 in the MSCC LP.3 cells. Input served as the positive control to validate antibody binding specificity, while IgG acted as the negative control to rule out nonspecific binding. (D) EP300 enrichment at Sox2 -associated loop target region on MSCC LP.3 gRNA-Control and gRNA- Smad4 cells detected by ChIP-qPCR. (E) H3K27ac enrichment at Sox2 -associated loop target region on MSCC LP.3 gRNA-Control and gRNA- Smad4 cell lines detected by ChIP-qPCR. Input served as the positive control to validate antibody binding specificity, while IgG acted as the negative control to rule out nonspecific binding. (F) The knockout efficiency of Ep300 in MSCC LP.3 cells with or without SMAD4 depletion by Western Blot, and the analysis of SMAD4 and SOX2 expression changes after Ep300 knockout in these cells. GAPDH served as the loading control. (G) Analysis of the changes in the frequency of Sox2 -associated loop by 3C-qPCR after Ep300 knockout in the MSCC LP.3 cell line with or without SMAD4 depletion. (H) Colony formation assay after the knock-out of the Sox2 -associated loop target regions in Smad4 and/or Ep300 knockout in the MSCC LP.3 cell line. Quantification of the relative gray value ratio was performed using ImageJ.

Article Snippet: After blocking with 5% non-fat milk for 1 hour at room temperature (RT), membranes were incubated overnight at 4 °C with primary antibodies against SMAD4 (1:1000, CST#38454), SOX2 (1:250, CST#14962), EP300 (1:500, Sigma-Merk 05-257), GAPDH (1:1000, CST#2118), or β-Actin (1:500, HANGZHOU FUDE BIOLOGICAL TECHNOLOGY CO., LTD., FD0060), followed by HRP-conjugated secondary antibodies (1:5000, Easy bio, BE0101 and BE0102).

Techniques: ChIP-sequencing, Binding Assay, Co-Immunoprecipitation Assay, Positive Control, Negative Control, Control, ChIP-qPCR, Knock-Out, Western Blot, Expressing, Colony Assay

Journal: bioRxiv

Article Title: A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

doi: 10.64898/2026.03.28.714793

Figure Lengend Snippet: (A) SOX2 -associated loops detected in Hi-C analysis of LUSC and adjacent normal samples. (B) Published ChIP-Seq data indicated the binding of EP300 and H3K27ac histone modification on SOX2 -associated loops detected in clinical LUSC samples. (C) RT-qPCR analysis of SMAD4 and SOX2 expression in SMAD4 knockout (gRNA- SMAD4 ) and non-knockout (gRNA-Control) in H2170. (D) Western Blot analysis of SMAD4 and SOX2 protein levels in SMAD4 knockout (gRNA- SMAD4 ) and non-knockout (gRNA-Control) in H2170. (E) SMAD4 expression levels and SOX2 expression levels at different time points following SMAD4 knockdown. (F) Colony formation assay in H2170 cell line with or without SMAD4 depletion and the quantification of the relative gray value ratio using ImageJ. (G) 3C-qPCR analysis of the relative loop frequency in H2170 cell lines with or without SMAD4 knockout. (H) Schematic diagram of the SOX2 -associated enhancer knockout strategy. (I) Western Blot analysis of SOX2 expression in the H2170 cells with or without the knockout of the enhancer of the SOX2 -associated loop. (J) Colony formation assay in the H2170 gRNA- SMAD4 cells with or without the knockout of the enhancer of SOX2 -assocaited loop, and the quantification of the relative gray value ratio using ImageJ. (K) Co-IP confirmed the interaction between EP300 and SMAD4 in the H2170 cells. Input served as the positive control to validate antibody binding specificity, while IgG acted as the negative control to rule out nonspecific binding. (L) Expression level of SMAD4 , EP300 , and SOX2 in clinical LUSC and normal samples. (M) The expression relevance between SMAD4 , EP300 , and SOX2 in LUSC samples.

Article Snippet: After blocking with 5% non-fat milk for 1 hour at room temperature (RT), membranes were incubated overnight at 4 °C with primary antibodies against SMAD4 (1:1000, CST#38454), SOX2 (1:250, CST#14962), EP300 (1:500, Sigma-Merk 05-257), GAPDH (1:1000, CST#2118), or β-Actin (1:500, HANGZHOU FUDE BIOLOGICAL TECHNOLOGY CO., LTD., FD0060), followed by HRP-conjugated secondary antibodies (1:5000, Easy bio, BE0101 and BE0102).

Techniques: Hi-C, ChIP-sequencing, Binding Assay, Modification, Quantitative RT-PCR, Expressing, Knock-Out, Control, Western Blot, Knockdown, Colony Assay, Co-Immunoprecipitation Assay, Positive Control, Negative Control

Journal: bioRxiv

Article Title: A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

doi: 10.64898/2026.03.28.714793

Figure Lengend Snippet: (A) DEGs regulated by remodeled chromatin loops indirectly regulated by SMAD4. (B) PRR19 -associated loops detected in BEAS-2B with or without SMAD4 KO cells and clinical LUSC samples. (C) Working model.

Article Snippet: After blocking with 5% non-fat milk for 1 hour at room temperature (RT), membranes were incubated overnight at 4 °C with primary antibodies against SMAD4 (1:1000, CST#38454), SOX2 (1:250, CST#14962), EP300 (1:500, Sigma-Merk 05-257), GAPDH (1:1000, CST#2118), or β-Actin (1:500, HANGZHOU FUDE BIOLOGICAL TECHNOLOGY CO., LTD., FD0060), followed by HRP-conjugated secondary antibodies (1:5000, Easy bio, BE0101 and BE0102).

Techniques: