binding buffer  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: An FGF7-FGFR2-KLF4 feedback loop sustains anti-inflammatory signaling in epithelial cells

doi: 10.64898/2026.03.20.711763

Figure Lengend Snippet: A) Volcano plots showing KLF4 affinity purification-mass spectrometry (KLF4 AP-MS) data from serum-starved HaCaT keratinocytes treated with FGF7 for 6 h or vehicle vs . the respective IgG control. Enriched prey proteins are located in the upper right quadrant, with KLF4 highlighted (N = 3, one-sided Student’s t -test, FDR<0-05). B) Volcano plot depicting FGF7-dependent changes in KLF4-associated proteins (KLF4 AP-MS data) under the same experimental conditions. p < 0.05 (two-sided Student’s t -test), filtering threshold ≥3 unique tryptic peptides per protein. C) Proteins, which also showed a significantly different abundance in the KLF4 interactome upon FGF7 treatment in an independent experiment. D) Schematic representation of the luciferase reporter vector harboring 4 KLF4 binding sites in the promoter. E) Luciferase activity in lysates of serum-starved HaCaT keratinocytes, stably transduced with lentiviruses including a luciferase reporter gene preceded by KLF4 response elements. Cells were treated for 8, 16 or 24 h with FGF7 or vehicle (N = 6). F) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing a luciferase reporter gene preceded by KLF4 response elements. Cells had been serum-starved and pre-treated for 2 h with the MEK1/2 inhibitor U0126 or vehicle, followed by a 6 h treatment with FGF7 or vehicle (N = 6). G) IL6 promoter cloning strategy showing the deletion of two KLF4 binding sites (BS) and the insertion of the IL6 promoter fragment into the firefly luciferase lentiviral vector. H) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing the IL6 promoter fragment with or without KLF4 BS in front of a luciferase gene. Cells were serum-starved and treated for 6 h with FGF7 or vehicle (N = 6). I) Luciferase activity in lysates of serum-starved HaCaT keratinocytes, stably transduced with lentiviruses containing the IL6 promoter fragment with or without KLF4 BS in front of a luciferase gene. Cells had been serum-starved and pre-treated for 3 h with FGF7 or vehicle and incubated for 6 h with poly(I:C), TNFα, or vehicle (N = 6). Data information: Graphs show mean and SD. Non-significant (ns), *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 (Mann-Whitney U test (D, G; normalized to respective control), or 2-way ANOVA with Bonferroni’s multiple comparisons test (E; H).

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Control, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Stable Transfection, Transduction, Cloning, Incubation, MANN-WHITNEY

Journal: bioRxiv

Article Title: An FGF7-FGFR2-KLF4 feedback loop sustains anti-inflammatory signaling in epithelial cells

doi: 10.64898/2026.03.20.711763

Figure Lengend Snippet: A) ChIP-seq data from the Gene Transcription Regulation Database (GTRD) showing the number of KLF4 binding sites (BS) in the promoter regions (−100 to +10 bp relative to the TSS) of FGF7-suppressed genes. B) RT-qPCR for IL6, RSAD2, ISG20 relative to RPL27 using RNA from serum-starved HaCaT keratinocytes or HPKs, which had been transfected with scrambled (scr) or KLF4 siRNA (N = 6; HPKs from two donors). C) Luciferase activity in lysates of HaCaT keratinocytes, stably transduced with lentiviruses containing the IL6 promoter fragment with or without KLF4 binding sites in front of a luciferase gene. Cells had been transfected with scrambled (scr) or KLF4 siRNA, serum-starved, and treated for 6 h with FGF7 or vehicle (N = 6). Data information: Graphs show mean and SD. Non-significant (ns), **P < 0.01, ****P < 0.0001 (Mann-Whitney U test (B, C)).

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: ChIP-sequencing, Binding Assay, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Stable Transfection, Transduction, MANN-WHITNEY

Journal: bioRxiv

Article Title: An FGF7-FGFR2-KLF4 feedback loop sustains anti-inflammatory signaling in epithelial cells

doi: 10.64898/2026.03.20.711763

Figure Lengend Snippet: A) Western blot of lysates from serum-starved HaCaT keratinocytes, transfected with scrambled (scr) or KLF4 siRNA mix and treated at 48 h post transfection with FGF7 or vehicle for 15 min. Graphs show densitometric quantification of KLF4, FGFR2 and total FRS2α band intensities normalized to the intensity of α-tubulin (upper panel) or p-FRS2α/FRS2α and p-ERK1/2/total ERK1/2 ratios (lower panels) (N = 3). B) RT-qPCR for DUSP6 and INHBA relative to RPL27 using RNA from serum-starved HaCaT keratinocytes, transfected with scr or KLF4 siRNA and treated at 48 h post transfection with FGF7 or vehicle for 6 h (N = 3). C, D) RT-qPCR for FGFR2 and FRS2A using RNA from serum-starved HaCaT keratinocytes or HPKs, transfected with scr or KLF4 siRNA (N = 3-6; HPKs from two donors). E) ChIP-seq data from the GTRD showing the number of KLF4 binding sites (BS) in the promoter regions (−500/−100 to +10/+50 bp relative to the TSS) of the FRS2A and FGFR1 - FGFR4 genes. F) RT-qPCR for FGFR2 and FRS2A using RNA from serum-starved HaCaT keratinocytes, incubated with membrane-permeable KLF4-TAT, FITC-TAT, or vehicle for 8 h (N = 6). Data information: Graphs show mean and standard deviation (SD). Non-significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t-test (A, normalized to respective control; C, D), one-way ANOVA with Bonferroni’s multiple comparisons test (F) or 2-way ANOVA with Bonferroni’s multiple comparisons test (B)).

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: Western Blot, Transfection, Quantitative RT-PCR, ChIP-sequencing, Binding Assay, Incubation, Membrane, Standard Deviation, Control

Journal: bioRxiv

Article Title: An FGF7-FGFR2-KLF4 feedback loop sustains anti-inflammatory signaling in epithelial cells

doi: 10.64898/2026.03.20.711763

Figure Lengend Snippet: A) Scheme depicting the FGF7–FGFR2b-ERK1/2-KLF4 signaling axis and its effect on inflammatory gene expression keratinocytes. The icons were obtained from BioRender. Werner, S. (2026) https://BioRender.com/kreppyv . B) Representative immunofluorescence stainings of normal human skin for FGFR2 (purple) and KLF4 (green). Scale bar: 20 μm.

Article Snippet: The beads were resuspended in 80 μl binding buffer, to which 9.21 μg of KLF4 antibody (Cell Signaling) or 9.21 μg of rabbit control IgG antibody (Merck, Darmstadt, Germany) was added.

Techniques: Gene Expression, Immunofluorescence

Journal: bioRxiv

Article Title: The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

doi: 10.64898/2026.02.24.707757

Figure Lengend Snippet: (A) Immuno-FISH analysis of wtAAV2-infected U2OS cells at an MOI of 5,000 vg/cell at 24 hpi where cellular DNA damage was induced by laser micro-irradiation. The location of the wtAAV2 genome was monitored by FISH using primers complementary to the wtAAV2 genome (green) and the DNA damage site was assessed using γH2AX staining (red). DAPI represents the nucleus (blue) with white dashed line demarcating the nuclear border. The white scale bar represents 10 microns. (B) Average of wtAAV2 signal intensity that colocalized with the γH2AX staining in multiple nuclei averaged over 10 microns of laser striping divided into 75 smaller bins. Data is presented as mean ± SEM of at least 10 independent laser micro-irradiated cells. (C) Results of the in-silico prediction analysis of host transcription factors binding sites that were enriched at wtAAV2-associated genomic region (computed using JASPAR informatics resource ). The most common consensus motifs (leftmost column) were compared with the p-value of their enrichment in the given regions (rightmost column). (D) Outcomes of in-silico analysis of KLF4 binding sites in the promoter regions of ribosomal genes that make up the nucleolus. The columns represent the number of KLF4 binding elements detected in each of the indicated gene promoters with the heatmap intensities depicting the strength of predicted KLF4 binding. (E) Analysis of the presence of KLF4 binding sites (assessed by KLF4 ChIP-seq in MCF7 cells) within the 10 kb windows spanning the promoters of ribosomal DNA genes (left; red heatmaps) compared with control sites on the host genome generated randomly (right; blue heatmaps). The profiles of the binding elements are shown on the line graph above each respective heatmap and was computed using deeptools on the Galaxy platform . (F) Schematic of the wtAAV2 genome with the promoters and respective KLF4 binding sites demarcated in the inset. The zoomed-in insets indicate the KLF4 binding sequences highlighted in grey and their positions on the wtAAV2 genome. (G) Immuno-FISH images of U2OS cells infected with wtAAV2 at an MOI of 5,000 vg/cell at 24 hpi showing the relative location of KLF4 (red) with that of the viral genome (green). The nucleus is demarcated by DAPI (blue stain) and the nuclear borders are marked by dashed white lines. The scale bar represents 10 microns. (H) Western blot analysis of the impact of wtAAV2 infection of 293T cells at the indicated timepoints on the global KLF4 levels (middle) and phosphorylated KLF4 at serine 245 (top blot). Tubulin levels serve as loading controls for the immunoblots.

Article Snippet: Primary antibodies used in this study and their respective dilutions used in western blots were: γH2AX (Abcam, ab11174, 1:1000); α-Tubulin (Millipore, 05-829, 1:5000); REP68/78 (IF11.8, 1:1000); PARP1 (BD Biosciences, 556494, 1:1000); KLF4 (Cell Signaling Technology, 4038S, 1:1000); PARylation (Cell Signaling Technology, 89190, 1:1000).

Techniques: Infection, Irradiation, Staining, In Silico, Binding Assay, ChIP-sequencing, Control, Generated, Western Blot

Journal: bioRxiv

Article Title: The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

doi: 10.64898/2026.02.24.707757

Figure Lengend Snippet: (A) ( top ) Schematic of wtAAV2 genome with the location of the HindIII site (blue) and NlaIII site (yellow) used for inverse PCRs indicated by forward (blue arrow) and reverse (yellow arrow) primers respectively. ( middle and bottom ) Representative UCSC genome browser tracks on human Chromosome 7 and Chromosome 4 of the indicated wtAAV2 variants identified using V3C-seq and compared with previously published KLF4 ChIP-seq in MCF7 cells and γH2AX in 293T cells . (B) Genome-wide localization of the wtAAV2 (top) and wtAAV2 ΔKLF4 (bottom) focused on the top 150 virus-associated HindIII fragments on the human genome. (C) Intersection of the genome coverage of the top 150 HindIII-localization sites of wtAAV2/wtAAV2 ΔKLF4 viruses, with the (D) statistical significance depicted by Jaccard analysis. Jaccard values represent the extent of overlap, with 0 indicating no overlap and 1 indicating complete overlap. The “permuted” samples are computed by intersecting the V3C-seq data with a randomly generated library of 150 fragments of 5 kb size across the human genome. (E) Results of the in-silico analysis using MEME and TOMTOM pipelines of the fragments that are represented in the distinct subsets in . The consensus sequences are presented in the left column and the corresponding transcription factors that have binding sites in the respective motifs are presented on the right columns. (F) Genome-wide profiling of wtAAV2 (left) and wtAAV2 ΔKLF4 (right) localization sites relative to all KLF4 binding sites identified by ChIP-seq within 100 kb bins. These heatmaps were computed using the deeptools resource on the Galaxy project server . (G) Venn diagrams showing the extent of overlap of the top 150 wtAAV2 localization sites on the human genome in the absence and presence of the PARP inhibitor Olaparib (labelled as iPARP). (H) Statistical analysis of the overlap was performed using Jaccard analysis (BEDtools suite ) using the same principle as described above.

Article Snippet: Primary antibodies used in this study and their respective dilutions used in western blots were: γH2AX (Abcam, ab11174, 1:1000); α-Tubulin (Millipore, 05-829, 1:5000); REP68/78 (IF11.8, 1:1000); PARP1 (BD Biosciences, 556494, 1:1000); KLF4 (Cell Signaling Technology, 4038S, 1:1000); PARylation (Cell Signaling Technology, 89190, 1:1000).

Techniques: ChIP-sequencing, Genome Wide, Virus, Generated, In Silico, Binding Assay

Journal: bioRxiv

Article Title: The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

doi: 10.64898/2026.02.24.707757

Figure Lengend Snippet: (A) Western blot analysis confirming KLF4 knockdown in U2OS cells transfected with two independent siRNAs targeting KLF4 mRNAs compared with siMock–transfected cells infected with wtAAV2 at an MOI of 5,000 vg/cell for 24 hours. Total cellular Tubulin levels were used as loading control. The right half represents quantification and the mean ± SEM of the KLF4 levels relative to Tubulin in three independent replicates. (B) Quantification of Rep68/78 transcripts relative to Actb transcripts in wtAAV2-infected U2OS cells (left) and 293T cells (right) at 24 hpi after knockdown for 24 hours. Data represents the mean ± SEM of three independent experiments with the statistical significance computed using t tests, ** representing p < 0.01, **** representing p < 0.0001. (C) Representative images of the impact of KLF4 knockdown on wtAAV2 localization to induced cellular sites of DNA damage evaluated using laser micro-irradiation coupled with Immuno-FISH in wtAAV2 infected U2OS cells at 24 hpi. The DNA damage sites were monitored by PARylation staining (red) and the viral genomes were tracked by Alexa-Fluor-488-labelled oligos complementary to the wtAAV2 genome (green). The nucleus was monitored by DAPI staining (blue) and the nuclear borders were demarcated by dashed white lines. The white scale bar represents 10 microns. (D) The profile of the intensity of wtAAV2 signal (green) that colocalized with DNA damage monitored by PARylation signal (red) in Immuno-FISH assays across 20-30 nuclei and averaged over the length of the laser micro-irradiated stripe by dividing into 90 bins. Data represents the mean ± SEM of the colocalized intensity at the computed site along the micro-irradiated stripe.

Article Snippet: Primary antibodies used in this study and their respective dilutions used in western blots were: γH2AX (Abcam, ab11174, 1:1000); α-Tubulin (Millipore, 05-829, 1:5000); REP68/78 (IF11.8, 1:1000); PARP1 (BD Biosciences, 556494, 1:1000); KLF4 (Cell Signaling Technology, 4038S, 1:1000); PARylation (Cell Signaling Technology, 89190, 1:1000).

Techniques: Western Blot, Knockdown, Transfection, Infection, Control, Irradiation, Staining

Journal: bioRxiv

Article Title: The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

doi: 10.64898/2026.02.24.707757

Figure Lengend Snippet: (A) Schematic of the wtAAV2 genome with the indicated KLF4 site mutation that was generated by site-directed mutagenesis. The grey highlighted sequence in the wtAAV2 region indicates the KLF4 binding sequence. (B) The impact of KLF4 binding element mutation on the viral genome titers produced by 293T cells was monitored using qPCR in three independent replicates. (C) The impact on KLF4 binding and (D) PARP1 binding on the wtAAV2 ΔKLF4 genome was measured using ChIP-qPCR with primers complementary to the P5 region of the viral genome. ChIP-qPCR data is presented as mean ± SEM of percent input from 3 independent replicates of 293T cell infection with statistical significance computed using t tests. P values represent statistical significance, with *** representing p < 0.001. (E) The impact of KLF4 binding site deletion on Rep68/78 gene expression was measured using RT-qPCR in U2OS cells (left) and 293T cells (right) at 24 hpi after infection with an MOI of 5,000 vg/cell. Data represents the mean ± SEM of three independent experiments with the statistical significance relative to wtAAV2 computed using t tests, ** representing p < 0.01 and **** representing p < 0.0001. (F) Impact of KLF4 site deletion on wtAAV2 localization to the cellular sites of DNA damage was evaluated using laser micro-irradiation coupled with Immuno-FISH in wtAAV2 infected U2OS cells at 24 hpi. The DNA damage site was monitored by PARylation staining (red) and the viral genomes were monitored by fluorophore-labelled oligos complementary to wtAAV2 (green). The nucleus was monitored by DAPI staining (blue) and the nuclear borders are demarcated by dashed white lines. The white scale bar represents 10 microns. (G) Profile of the intensity of wtAAV2 signal (green) that colocalizes with DNA damage monitored by PARylation signal (red) in Immuno-FISH assays across at least 10 nuclei and averaged over the length of the laser micro-irradiated stripe. Data represents the mean ± SEM of the colocalized intensity at the computed site along the micro-irradiated stripe divided into 75 bins.

Article Snippet: Primary antibodies used in this study and their respective dilutions used in western blots were: γH2AX (Abcam, ab11174, 1:1000); α-Tubulin (Millipore, 05-829, 1:5000); REP68/78 (IF11.8, 1:1000); PARP1 (BD Biosciences, 556494, 1:1000); KLF4 (Cell Signaling Technology, 4038S, 1:1000); PARylation (Cell Signaling Technology, 89190, 1:1000).

Techniques: Mutagenesis, Generated, Sequencing, Binding Assay, Produced, ChIP-qPCR, Infection, Gene Expression, Quantitative RT-PCR, Irradiation, Staining

Journal: bioRxiv

Article Title: The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

doi: 10.64898/2026.02.24.707757

Figure Lengend Snippet: (A) Western blot analysis of the impact of wtAAV2 infection of 293T cells at an MOI of 5,000 vg/cell at 24 hpi. The PARP1 and PARylation levels were measured with the respective antibodies and Tubulin was used as loading control. The figure on the left is a representative immunoblot and the right shows the average of PARylation levels relative to Tubulin in 3 independent biological replicates of viral infection. Error bars represent SEM of PARylation to Tubulin ratios. (B) RT-qPCR analysis of Rep 68/78 transcript levels in wtAAV2 infected 293T cells in the presence of Olaparib (PARPi). The corresponding impact on (C) KLF4 binding (left) and PARP1 binding (right) at P5 was measured using ChIP-qPCR analysis. Statistical analysis was performed with a t test, ** representing p < 0.01. (D) Impact of Olaparib treatment (PARPi) on wtAAV2 localization to the cellular sites of DNA damage was evaluated using laser micro-irradiation coupled with Immuno-FISH in wtAAV2 infected U2OS cells at 24 hpi. The DNA damage site was monitored by PARylation staining (red) and the viral genomes were monitored by fluorophore-labelled oligos (green). The nucleus was monitored by DAPI staining (blue) and the nuclear borders are demarcated by dashed white lines. (E) The profile of wtAAV2 colocalizing with PARylation signal across multiple micro-irradiated nuclei was plotted in 75 discrete intervals along the DDR site. Data is presented as mean ± SEM of the colocalized intensity in at least 10 nuclei. (F) PARP1 western blot in wild-type and PARP1-deficient U2OS cells demonstrating knockout of the PARP1 protein with Tubulin as loading control. (G) RT-qPCR of Rep68/78 transcripts in PARP1-deficient U2OS cells that were complemented with the indicated PARP1 expression vectors for 24 hours before being infected with wtAAV2 for 24 hpi at an MOI of 5,000 vg/cell. Data is presented as mean ± SEM of expression relative to Actb in three independent replicates, with statistical significance computed using t tests, * representing p < 0.05, **** representing p < 0.0001 and ns denoting no statistical significance. (H) The protein stability of PARP1 during ectopic expression of PARP1-deficient cells was monitored by western blots 24 hours after transient transfection with the expression vector, and the mean ± SEM of three independent replicates of PARP1 to Tubulin ratios are presented on the top. Representative western blots are shown in the bottom.

Article Snippet: Primary antibodies used in this study and their respective dilutions used in western blots were: γH2AX (Abcam, ab11174, 1:1000); α-Tubulin (Millipore, 05-829, 1:5000); REP68/78 (IF11.8, 1:1000); PARP1 (BD Biosciences, 556494, 1:1000); KLF4 (Cell Signaling Technology, 4038S, 1:1000); PARylation (Cell Signaling Technology, 89190, 1:1000).

Techniques: Western Blot, Infection, Control, Quantitative RT-PCR, Binding Assay, ChIP-qPCR, Irradiation, Staining, Knock-Out, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

doi: 10.64898/2026.02.24.707757

Figure Lengend Snippet: (A) Schematic of the rAAV genomes showing the location of the insertion of the KLF4 binding element dimer sequences (represented by grey highlights). (B) Impact of rAAV2/rAAV2 KLF4 transduction on 293T cells transduced at an MOI of 5,000 vg/cell at 24 hpi. The figure demonstrates representative FACs plots from 3 independent experiments in live cells identified by forward and side-scatter analysis followed by assessment for GFP positivity. The percent positive cells are shown in the inset. (C) Average mean fluorescence intensity of GFP expression in 293T cells transduced with the indicated rAAV vectors for 24 hpi at an MOI of 5,000 vg/cell. (D) These cell populations were assessed for GFP positivity in live cells using FACS analysis. Data is presented as mean ± SEM of cells from at least three independent transduction experiments with statistical analysis performed using t tests. P values are computed as: ** representing p < 0.01 and **** representing p < 0.0001. (E) Representative Immuno-FISH images of U2OS cells transduced with the indicated rAAV vectors at an MOI of 5,000 vg/cell for 24 hpi followed by co-staining for vector genomes (green), PARylation (red) and DAPI (blue) to demarcate the nuclei. The white dashed lines indicate the nuclear borders and the white line represents 10 micrometers. White arrows in the nuclei indicate regions of colocalization between the vector genome and PARylation.

Article Snippet: Primary antibodies used in this study and their respective dilutions used in western blots were: γH2AX (Abcam, ab11174, 1:1000); α-Tubulin (Millipore, 05-829, 1:5000); REP68/78 (IF11.8, 1:1000); PARP1 (BD Biosciences, 556494, 1:1000); KLF4 (Cell Signaling Technology, 4038S, 1:1000); PARylation (Cell Signaling Technology, 89190, 1:1000).

Techniques: Binding Assay, Transduction, Fluorescence, Expressing, Staining, Plasmid Preparation