rabbit anti yap1  (Novus Biologicals)

Journal: Journal of Translational Medicine

Article Title: TROP2 confers resistance to oxidative stress-induced cancer cell death through YAP/HMOX1 signaling

doi: 10.1186/s12967-026-07955-z

Figure Lengend Snippet: TROP2 suppression sensitized NSCLC to RSL3-induced ferroptosis in vivo. ( A ) Schematic diagram of the in vivo experimental timeline. Nude mice were subcutaneously injected with control (shCON) or TROP2-knockdown (shTROP2) PC9 cells, followed by intraperitoneal injection of RSL3 (5 mg/kg) or vehicle every other day. ( B ) Representative photographs of dissected subcutaneous tumors from each treatment group at the endpoint of the study. ( C ) Final tumor weights from each group at the endpoint. Data are presented as mean ± SD ( n = 6). ( D ) Tumor growth curves showing tumor volume over time for the four experimental groups. Data are presented as mean ± SEM ( n = 6 mice per group). ( E ) Representative IHC images of tumor sections stained for TROP2, YAP1, HMOX1, ACSL4, and SLC7A11 from the indicated groups. Scale bar, 100 μm. Data are presented as mean ± SD ( n = 6, C) or mean ± SEM ( n = 6 mice per group, D ). Statistical significance for differences among groups was determined by one-way ANOVA followed by Tukey’s post hoc test (C, and for the final time point in D). ∗ P < 0.05, ∗∗ P < 0.01

Article Snippet: After that, the membranes were blocked in 5% fat-free milk for 1 h. The membranes were incubated with the primary antibodies including TROP2 (1:1000, cat #47866, CST), GPX4 (1:1000, cat #AF7020, Beyotime), SLC7A11 (1:1000, cat #AF7992, Beyotime), ACSL4 (1:1000, cat #AG1908, Beyotime), HMOX1 (1:1000, cat #AG2181, Beyotime), YAP1 (1:1000, cat #14074, CST), P-YAP1 (1:1000, cat #13619, CST), LATS1 (1:1000, cat #3477, CST), p-LATS1 (1:1000, cat #9157, CST), Histone3 (1:1000, cat #9715, CST),α-Tubulin (1:1000, cat #2144, CST) at 4 °C overnight and the secondary antibodies at room temperature for 2 h. Protein bands were visualized using the ECL (Bio-Rad, Hercules, CA, USA).

Techniques: In Vivo, Injection, Control, Knockdown, Staining

Journal: Journal of Translational Medicine

Article Title: TROP2 confers resistance to oxidative stress-induced cancer cell death through YAP/HMOX1 signaling

doi: 10.1186/s12967-026-07955-z

Figure Lengend Snippet: TROP2 confers ferroptosis resistance by inactivating YAP to suppress HMOX1 transcription. ( A ) YAP1 mRNA levels in H292 and PC9 cells with TROP2 knockdown, as determined by qRT-PCR. ( B ) YAP1 mRNA levels in PC9 and A549 cells with TROP2 overexpression, as determined by qRT-PCR. ( C ) Representative IF images showing enhanced nuclear localization of YAP (green) in PC9 cells upon TROP2 knockdown. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( D ) Representative IF images showing suppressed nuclear localization of YAP (green) in PC9 cells upon TROP2 overexpression. Scale bar, 50 μm. ( E ) Western blot analysis of YAP protein levels in nuclear and cytoplasmic fractions of PC9 and H292 cells with or without TROP2 knockdown. ( F ) Quantitative analysis (bar graph) of the bands was shown adjacent to the representative blots (from E). ( G, H ) Cell viability assessed by CCK-8 assay in PC9 and H292 cells with TROP2 knockdown, with or without concomitant YAP inhibition, following RSL3 treatment. ( I,J ) Measurement of cellular GSH levels in PC9 and H292 cells under the indicated conditions. ( K, L ) Measurement of cellular MDA levels in PC9 and H292 cells under the indicated conditions. Data are presented as mean ± SD ( n = 3). Statistical significance was determined by two-tailed unpaired Student’s t-test ( A , B , F ); two-way ANOVA followed by Sidak’s post hoc test or three-way ANOVA followed by Sidak’s post hoc test ( G - L ). ∗ P < 0.05, ∗∗ P < 0.01

Article Snippet: After that, the membranes were blocked in 5% fat-free milk for 1 h. The membranes were incubated with the primary antibodies including TROP2 (1:1000, cat #47866, CST), GPX4 (1:1000, cat #AF7020, Beyotime), SLC7A11 (1:1000, cat #AF7992, Beyotime), ACSL4 (1:1000, cat #AG1908, Beyotime), HMOX1 (1:1000, cat #AG2181, Beyotime), YAP1 (1:1000, cat #14074, CST), P-YAP1 (1:1000, cat #13619, CST), LATS1 (1:1000, cat #3477, CST), p-LATS1 (1:1000, cat #9157, CST), Histone3 (1:1000, cat #9715, CST),α-Tubulin (1:1000, cat #2144, CST) at 4 °C overnight and the secondary antibodies at room temperature for 2 h. Protein bands were visualized using the ECL (Bio-Rad, Hercules, CA, USA).

Techniques: Knockdown, Quantitative RT-PCR, Over Expression, Staining, Western Blot, CCK-8 Assay, Inhibition, Two Tailed Test

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