rabbit anti yap1  (Novus Biologicals)

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: Stutter cysteine is sufficient to relocalize YAP1 in transfected HeLa cells. (A and B) Representative images and magnified insets of HeLa cells transfected with mCherry-K5, EGFP-K14, and untagged WT and mutant K15. The mCherry signal is autofluorescence, and YAP1 is visualized through indirect immunostaining. Yellow dashed lines outline nuclei, and green dashed line outline the cell peripheries. Scale bar, 10 µm. (C) Scatter plots representing three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, ***P = 0.0002 and 0.0003, **P = 0.0083.

Article Snippet: Fixed cells were washed, permeabilized for 5 min in 0.1% Triton X-100, and then incubated for 1 h with rabbit anti-YAP1 antibody (14074S; Cell Signaling Technology), followed by 1-h incubation with an Alexa Fluor–conjugated secondary antibody, and counterstained with DAPI.

Techniques: Transfection, Mutagenesis, Immunostaining, Standard Deviation, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: Stutter cysteine is sufficient to relocalize YAP1 in differentiating mouse skin keratinocytes. (A) Representative micrographs of Krt14 WT/null keratinocytes in primary culture transfected with EGFP-K14, EGFP-K15, or EGFP-K15 A351C, with and without 1.2 mM CaCl 2 to induce differentiation. YAP1 is visualized through immunofluorescence. “Merge” panels display autofluorescent EGFP (green) and YAP1 (magenta). Arrowheads indicate EGFP-positive keratinocytes. Scale bar, 10 µm. (A′) Magnified insets of nuclei with YAP1 indirect immunofluorescence from . Arrowheads indicate EGFP-positive keratinocytes. Scale bar, 10 µm. (B) Scatter plots representing three independent replicates, pooled, displaying mean ± SD. Dots represent a sc. Comparisons were made using unpaired t tests with Welch’s correction. ****P < 0.0001, **P = 0.0058 and 0.0014. (C) Multiplexed immunostaining for K15 (green) and YAP1 (red). Nuclei are counterstained with DAPI (blue). A dashed line specifies the basal lamina. epi, epidermis; derm, dermis. Scale bar, 50 µm. Arrowheads depict region with strong K15 and nuclear YAP1 staining. (D) XY scatter plot is comparing nuclear YAP1 intensity (arbitrary units) vs. K15 intensity (arbitrary units). Each dot represents a single basal keratinocyte. Equation for the simple linear regression of the comparison is displayed, showing a positive correlation. Regression is significantly nonzero (P < 0.0001).

Article Snippet: Fixed cells were washed, permeabilized for 5 min in 0.1% Triton X-100, and then incubated for 1 h with rabbit anti-YAP1 antibody (14074S; Cell Signaling Technology), followed by 1-h incubation with an Alexa Fluor–conjugated secondary antibody, and counterstained with DAPI.

Techniques: Transfection, Immunofluorescence, Immunostaining, Staining, Comparison

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: Analyses of keratin and 14-3-3 protein interactions (complement to ). (A–C′) Lollipop plots displaying the 14-3-3-Pred “consensus score” for all serines and threonines in (A) human KRT14 ; (A′) mouse Krt14 ; (B) human KRT5 ; (B′) mouse Krt5 ; (C) human KRT15 ; (C′) mouse Krt15 . Lollipop height and color both scale to consensus score magnitude. The two residues with the largest positive consensus score are labeled. (D) Lollipop plot illustrates identified phosphorylation sites on K14 protein exhibiting a localization score >0.6 in human N-TERT keratinocytes in culture. The relative occupancy percentage for each phosphorylation site was calculated by dividing the phosphopeptide’s intensity by total intensity of unique K14 peptides. This calculation, shown here as relative phosphorylation site occupancy (%), was performed only for phosphopeptides that are unique to K14 without miscleavages (S44, S281, S435, S437). (D′) Spectra of the phosphopeptides corresponding to the S39 (left) and S44 (right) sites. “Phos” conveys phosphorylation, and “CAM” stands for carbamidomethyl. (E) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm.

Article Snippet: Fixed cells were washed, permeabilized for 5 min in 0.1% Triton X-100, and then incubated for 1 h with rabbit anti-YAP1 antibody (14074S; Cell Signaling Technology), followed by 1-h incubation with an Alexa Fluor–conjugated secondary antibody, and counterstained with DAPI.

Techniques: Labeling, Phospho-proteomics, Transfection, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: SCRAPS motif and the stutter cysteine are required to interconvert YAP1 interaction between K14 and K15. (A) Representative co-immunoprecipitations of HeLa cells transfected with untagged K5 and EGFP-tagged WT K14, K15, and mutant K15. Pull-down of endogenous 14-3-3σ is depicted. (B) Differential levels of WT K15, relative to WT K14 or mutant K15, were observed in the soluble fraction. Transfection of keratin(s) did not affect endogenous 14-3-3σ levels. WB intensity was normalized to a loading control (histone H3). Dots represent three independent replicates, displaying mean and standard error. Comparisons were made using one-way ANOVA, ****P < 0.0001, ns = not significant. (B′) Endogenous 14-3-3σ pulls down more efficiently with WT K14 and mutagenized K15, as compared to WT K15. Equation to calculate 14-3-3σ pull-down efficiency is described in Materials and methods. Dots represent three independent replicates. Comparisons were made using one-way ANOVA, **P = 0.0055, *P = 0.0237, ns = not significant. (C) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and EGFP-tagged WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm. (D) Scatter plots representing YAP1-14-3-3σ PLA punctae counted per cell in three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, **P = 0.0033, ns = not significant. (E) Transcription of a YAP1 target gene, CYR61 , as measured via RT-qPCR in HeLa cells transfected with untagged K5 and WT and mutant K14 or K15. Dots represent six independent replicates. Comparisons were made using Mann–Whitney tests, **P = 0.0033, ns = not significant. Source data are available for this figure: .

Article Snippet: Fixed cells were washed, permeabilized for 5 min in 0.1% Triton X-100, and then incubated for 1 h with rabbit anti-YAP1 antibody (14074S; Cell Signaling Technology), followed by 1-h incubation with an Alexa Fluor–conjugated secondary antibody, and counterstained with DAPI.

Techniques: Transfection, Mutagenesis, Control, Standard Deviation, MANN-WHITNEY, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: Molecular analyses of Krt14 C373A/null transgenic mouse skin (complement to ). (A and A′) K14 and (A′) K15 dot blots of whole tail skin lysate from 8-wk-old male Krt14 C373A/WT and Krt14 C373A/null littermates. For K14 immunoblotting, 7.5, 5, 2.5, 1, and 0.5 µg of lysate were loaded onto a membrane. For K15 immunoblotting, 5, 2.5, 1, 0.75, and 0.5 µg of lysate were loaded onto a membrane. Histone H3 was utilized as a loading control. Mean of K14 and K15 dot-blot MGV was normalized to histone H3. Comparisons were made using Mann–Whitney tests. **P = 0.0025. (B) Growth curve of mean weight of male and female Krt14 C373A/WT and Krt14 C373A/null littermates. Error bars represent SD. (C) Transcription of a YAP1 target gene, Cyr61 , as measured via RT-qPCR in whole tail skin lysate from 8-wk-old male and female Krt14 C373A/WT and Krt14 C373A/null littermates. Dots represent three biological replicates. Comparisons were made using Mann–Whitney tests. *P < 0.005.

Article Snippet: Fixed cells were washed, permeabilized for 5 min in 0.1% Triton X-100, and then incubated for 1 h with rabbit anti-YAP1 antibody (14074S; Cell Signaling Technology), followed by 1-h incubation with an Alexa Fluor–conjugated secondary antibody, and counterstained with DAPI.

Techniques: Transgenic Assay, Western Blot, Membrane, Control, Dot Blot, MANN-WHITNEY, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: K15 overrepresentation alters defining attributes of basal layer keratinocytes in vivo . (A) Krt14 C373A/WT and Krt14 C373A/null (arrow) littermates at P5. (B) H&E staining performed on back skin harvested from P5 Krt14 C373A/WT and Krt14 C373A/null littermate pups. Scale bar, 50 µm. (B′) Magnified insets of H&E-stained back skin in . Scale bar, 10 µm. (C) Epidermal thickness (µm) and hair follicle depth (µm) in back skin harvested from P5 Krt14 C373A/WT and Krt14 C373A/null littermate pups. Comparisons were made using Mann–Whitney tests. ****P < 0.0001. (C′) Mean cytoplasmic area of basal keratinocytes with standard deviation; dots represent scs. Comparisons were made using Mann–Whitney tests. *P = 0.0286. Basal cell density was calculated by mean cells/µm of basal lamina with standard deviation; dots represent individual micrographs from tail skin harvested from four littermate animals each. Comparisons were made using Mann–Whitney tests. *P = 0.014. (D) H&E staining performed on tail tissue harvested from 8-wk-old Krt14 C373A/WT and Krt14 C373A/null tail tissue. Scale bar = 50 µm. (E) YAP1 and K15 immunofluorescence performed on 8-wk-old Krt14 C373A/WT and Krt14 C373A/null littermate tail skin. Merge panel displays DAPI counterstain (blue), YAP1 (green), and K15 (red). Arrows emphasize nuclei of suprabasal keratinocytes, showing YAP1-negative nuclei in Krt14 C373A/WT tail skin and YAP1-positive nuclei in Krt14 C373A/null tail skin. A dashed line specifies the basal lamina. epi, epidermis; derm, dermis. Scale bar, 50 µm. (F) Representative transmission electron microscopy micrographs of 8-wk-old Krt14 C373A/WT and Krt14 C373A/null basal keratinocytes in ear skin. Nu, nucleus; kf, keratin filaments; arrows, hemidesmosomes; *, areas devoid of keratin filaments. Scale bar, 800 nm. (F) Ki67 immunofluorescence performed on 8-wk-old Krt14 C373A/WT and Krt14 C373A/null littermate tail skin. Merge panel displays Ki67 (green) with DAPI counterstain (red). Arrows indicate Ki67-positive basal nuclei. A dashed line specifies the basal lamina. epi, epidermis; derm, dermis. Scale bar, 50 µm. (G) Mean of the basal lamina processed/distance covered to represent basal lamina convolution, displayed with standard deviation. Dots are individual micrograph images. Comparisons were made using Mann–Whitney tests. ***P = 0.0009. (G′–H′) Mean of the Ki67 MIV for individual basal keratinocyte nuclei, displayed with standard deviation. Percentages of cells above the visual threshold for Ki67 positivity (green dashed line). Comparisons were made using Mann–Whitney tests. ****P < 0.0001.

Article Snippet: Fixed cells were washed, permeabilized for 5 min in 0.1% Triton X-100, and then incubated for 1 h with rabbit anti-YAP1 antibody (14074S; Cell Signaling Technology), followed by 1-h incubation with an Alexa Fluor–conjugated secondary antibody, and counterstained with DAPI.

Techniques: In Vivo, Staining, MANN-WHITNEY, Standard Deviation, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: The regulatory landscape of optic fissure closure in the vertebrate eye

doi: 10.64898/2026.02.11.705341

Figure Lengend Snippet: ( A-D ) Number of peaks and highest match de novo motifs identified by HOMER for each context specific category. Only motifs with a percentage of target to background ratio > 1.5 were included. ( E-H ) Histograms showing motif occurrence distribution around the centre of peaks corresponding to analysis in A-D. Lines without observable enrichment are translucent. Motif consensus sequences and their likely TF identities are shown below and heights correlate to the frequency of base occurrence. ( I ) YAP1 immunofluorescence during chicken OFC at HH30 showed highest signal (arrows) in the edges of the fusing OFM and in the fused margins. In contrast, low signal was detected in dorsal tissue. Abbreviations : RPE, retinal pigmented epithelium, NR, neural retina; POM, periocular mesenchyme.

Article Snippet: Immunofluorescence of YAP1 was performed using methods described previously , but using rabbit anti-human YAP1 antibody (D8H1X, Cell Signalling Technology) diluted 1:50 in block solution (PBS, 0.1% BSA, 0.05% Triton X-100) and incubated overnight at 4°C, and detected with Alexa-fluor 488nm goat anti-rabbit secondary antibody (1:1000 dilution; A11037, Thermo Fisher) and DAPI (1:1000 dilution; D3571, Thermo Fisher).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: The regulatory landscape of optic fissure closure in the vertebrate eye

doi: 10.64898/2026.02.11.705341

Figure Lengend Snippet: (A) Shared sequence similarities for homeodomain motifs LHX2, VAX1, ALX1 and LHX2. ( B ) TPM plot showing expression patterns of TEAD1 , TEAD3 , TEAD4 and YAP1 from the RNA-seq analysis . (C) TPM of transcription factors expression levels from the RNA-seq data. Only values for transcription factor genes with a motif enrichment indicated by HOMER were plotted.

Article Snippet: Immunofluorescence of YAP1 was performed using methods described previously , but using rabbit anti-human YAP1 antibody (D8H1X, Cell Signalling Technology) diluted 1:50 in block solution (PBS, 0.1% BSA, 0.05% Triton X-100) and incubated overnight at 4°C, and detected with Alexa-fluor 488nm goat anti-rabbit secondary antibody (1:1000 dilution; A11037, Thermo Fisher) and DAPI (1:1000 dilution; D3571, Thermo Fisher).

Techniques: Sequencing, Expressing, RNA Sequencing