anti arhgap29 antibody  (Novus Biologicals)

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: General characteristics of Arhgap29 −/− mice. ( A ) Gross image of the E15.5 mice. ( B ) Image of the head of the P0 mice (the black dashed box indicates the mandible). The penetrance of mandibular anomalies in Arhgap29 −/− mice is 30% (3/10). ( C ) Image of the palate of the E17.5 mice. The black dashed line indicates the cleft palate with 31.25% penetrance (5/16). ( D ) Image of the forelimb digits of the E14.5 mice. The black arrow indicates ectrodactyly with 96.67% penetrance (29/30).

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: ( A ) Three-dimensional reconstruction images of micro-CT scanning of WT and Arhgap29 deletion specimens. The white dashed line indicates the outline of the collapsed skull of the Arhgap29 −/− mice. FR, frontal bone; PA, parietal bone; IP, interparietal bone; M, mandible; co, coronal suture; sa, sagittal suture; la, lambdoidal suture. ( B ) The quantitative analysis of the cranium and mandible showed that the average bone surface area, bone volume, and bone mineral content were reduced to varying degrees in both the cranium and mandible of Arhgap29 −/− mice compared with WT mice. BV, bone volume; BS, bone surface; BMC, bone mineral content. All data: *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Micro-CT

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Alcian blue and Alcian blue/alizarin red staining results of cartilage and bone in the craniofacial region and limbs of WT and Arhgap29 −/− mice. ( A ) Alcian blue staining was performed on Meckel’s cartilage during three developmental stages: E13.5, E14.5, and P0. ( B ) Alcian blue/alizarin red staining of cranial and facial bones at P0. ( C ) Alcian blue/alizarin red staining of the mandible at P0. ( D ) Alcian blue/alizarin red staining of the whole skeleton of mice. ( E ) Alcian blue/alizarin red staining of the front limbs of mice; the Arhgap29 −/− mice have ectrodactyly.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Histological characteristics of Meckel’s cartilage. The results of H&E staining of Meckel’s cartilage in E13.5 ( A ), E15.5 ( B ), and E17.5 ( C ) mice, along with Alcian blue staining of Meckel’s cartilage in P0 mice ( D ). ( A – C ) illustrate the coronal section staining results of the mouse head, demonstrating the delayed hypertrophy of Meckel’s chondrocytes in Arhgap29 −/− mice. ( D ) presents the axial section staining results of the mouse head, which reveal the delayed degeneration of Meckel’s cartilage. The red dashed line indicates Meckel’s cartilage (MC); the mandible is labeled as M. Three mice from each group were chosen for every time period.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Experimental results of osteogenesis in mandibular tissue. ( A , B ) Von Kossa staining of mandibular tissue sections from E17.5 mice. ( C ) ALP staining of mandibular tissue sections from E17.5 WT mice. ALP-positive cells (shown in blue) are distributed on the surface of the bone matrix. ( D ) Immunohistochemical staining results of mandibular tissue sections from E17.5 WT mice. Arhgap29 -positive cells (shown in brown-yellow) are located on the surface of the bone matrix. (The red arrow indicates osteoblasts).

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Staining, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Experimental results of osteoclast activity in mouse mandibular tissue. ( A , B ) TRAP staining of mandibles in E17.5 WT and Arhgap29 −/− mice (osteoclasts are stained red, as indicated by the black arrows). ( C ) Staining of osteoclast marker TRAP in mandibles of WT mice at E17.5. ( D ) Immunohistochemical staining of osteoclasts in mandibles of WT mice at E17.5 (with positive cells indicated in brownish-yellow).

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Activity Assay, Staining, Marker, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: Analysis of transcriptome sequencing results for E17.5 mandibular tissue. ( A ) Volcano plot of differentially expressed genes. ( B ) GO classification annotation and enrichment analysis. ( C ) KEGG classification annotation and pathway enrichment analysis. ( D ) Heatmap of differentially expressed gene clustering in calcium signaling pathway. ( E ) Heatmap of differentially expressed gene clustering in cell differentiation. ( F ) qPCR validation of calcium signaling pathway-related molecules. ( G ) qPCR validation of cell differentiation-related molecules. (The internal reference gene used was Gapdh , and the relative expression of the Arhgap29 −/− group was calculated based on the gene expression levels of the WT group. Statistical analyses of differences were performed using a t -test. * indicates a statistically significant difference between groups. *** p < 0.001; ** p < 0.01; ns indicates no significant difference.)

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: Sequencing, Cell Differentiation, Biomarker Discovery, Expressing, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Arhgap29 Deficiency Directly Leads to Systemic and Craniofacial Skeletal Abnormalities

doi: 10.3390/ijms26104647

Figure Lengend Snippet: In vitro cell experiment results. ( A ) qPCR assay for osteoblast markers in cells 3 days after they were induced to differentiate. ( B , C ) ALP staining of WT and si Arhgap29 cells 7 days after they were induced to differentiate. ( D , E ) ARS staining of WT and si Arhgap29 cells after they were induced to differentiate for 14 days. ( F ) Quantitative analysis of alkaline phosphatase staining in cells ( n = 5). ( G ) Quantitative analysis of alizarin red staining in cells ( n = 5). All data: *** p < 0.001.

Article Snippet: After dewaxing and rehydration, the sections were incubated with the Arhgap29 primary antibody (sc-377022, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with a secondary antibody (Bioss, Beijing, China) for 30 min at 37 °C.

Techniques: In Vitro, Staining

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 2. Gene expression analysis un- covers dysregulation of members of the ARHGAP gene family in U251 cells, and sta- ble silencing of ARHGAP12 and ARHGAP29 in U251 cells exerts distinct cytoskeletal re- arrangements (A and B) Representative immunofluorescence images of ARHGAP12 and ARHGAP29 expres- sion, untreated and after exposure to the GSK-3 inhibitor BIO (A), with quantification by total fluo- rescence (B). Student’s t test, *p < 0.05. Scale bar: 10 mm. Data presented as mean ± SEM. (C) Measurement of cellular localization showed loss of nuclear expression of both ARHGAP12 and ARHGAP29 following exposure to BIO. (D) Representative bright-field micrographs of collagen-embedded U251 spheroids immuno- stained for either ARHGAP12 or ARHGAP29 (brown) and counterstained with hematoxylin. Cytoplasmic labeling of ARHGAP12 in the spheroid core became more pronounced after BIO treatment (black arrowheads). For ARHGAP29, cytoplasmic and membranous labeling was noted, especially on the spheroid periphery and on migratory cells, which was reduced after treatment with BIO (red arrowheads). Scale bar: 50 mm. Data presented as median. (E) Stable gene silencing of ARHGAP12 (A12 kd) and ARHGAP29 (A29 kd) in U251 cells was confirmed by western blot. (F) Representative immunofluorescence of U251 cells with stable ARHGAP12 and ARHGAP29 kd showing morphological changes and cytoskeletal rearrangement in U251 cells in 2D monolayers. Scale bar: 100 mm. (G) Time-lapse microscopy of U251 cells with kd of the 2 different ARHGAPs showed distinct cellular morphological characteristics compared to control cells. Scale bar: 200 mm. (H) In 3D spheroid assays, over 72 h, shorter cell protrusions consisting of rounded cells for the ARHGAP29 kd and protrusions consisting of in- terconnected, elongated cells became evident. Scale bar: 100 mm. (I) 3D invasion assays highlight cellular features and morphological changes of migrating cells after ARHGHAP29 and ARHGAP12 kd. Scale bar: 200 mm.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Gene Expression, Expressing, Staining, Labeling, Western Blot, Time-lapse Microscopy, Control

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 3. Stable silencing of ARHGAP12 or ARHGAP29 induces changes in the number of appendages emanating from spheroids and distance traveled away from spheroids by individual migratory cells Using Cloudbuster software,26 ARHGAP12 and ARHGAP29 kd cell spheroids were analyzed at time point 0 and at 48 h. (A) In U87 cells, ARHGAP29 kd spheroids showed reduced length of protrusions in comparison to ARHGAP12 (one-way ANOVA, p = 0.0002) but no difference in the number of extensions after 48 h (one-way ANOVA, p > 0.05). (B) For U251, a similar effect was seen after ARHGAP12 kd in the length of cellular protrusions compared to ARHGAP29 kd (p = 0.0016) after 48 h. Data presented as median, interquartile range. (C) Representative reconstructed spheroids and migratory cells treated with a non-target control, ARHGAP29 kd, or ARHGAP12 kd. Insets high- light a selected region of an individual spheroid with visible extensions and individual cells (white arrowheads) with (left to right) a non-target (NT) control spheroid, ARHGAP29 kd, and ARHGAP12 kd.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Software, Comparison, Control

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 4. ARHGAP transcription is regu- lated in part by GSK-3 signaling via b-cate- nin translocation (A) Immunofluorescence labeling with various markers (ARHGAP12, ARHGAP29, b-catenin, and CD44) of U251 cells treated with the GSK-3 in- hibitor BIO. ICG001 and inhibitor combination re- veals that ARHGAP12 and ARHGAP29 protein levels are altered after treatment with BIO and not affected by treatment with ICG001 alone or the combination treatment (n = 3/group). Scale bar: 200 mm. (B and C) Significant differences (mean ± SEM) in (B) CD44 expression and (C) b-catenin expression in cell populations treated with the GSK-3 inhibitor BIO, ICG001, or ICG001 in combination with the GSK-3 inhibitor BIO were observed (n = 3). Data presented as mean ± SEM. (D) Representative images from time-lapse mi- croscopy of U251 cells, showing changes in the position of cells over a 24-h period in untreated, BIO-treated, ICG001-treated, and combination treatment groups. Scale bar: 200 mm (E) Representative plots demonstrating the nega- tive effect of the GSK inhibitor BIO on cell migra- tion, with no effect of the inhibitor ICG001 and no effect after combination treatment. (F and G) Significant differences (mean ± SEM) in (F) ARHGAP12 and (G) ARHGAP29 expression in cell populations treated with the GSK-3 inhibitor BIO, ICG001, or ICG001 in combination with the GSK-3 inhibitor BIO were observed (n = 3). Data presented as mean ± SEM. Post hoc Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Translocation Assay, Labeling, Expressing

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 6. Intracranially injected ARHGAP12 and ARHGAP29 kd cells induce tumors with altered morphological features (A–C) Representative mouse brain tissue sections of intracranial tumors from non-target control, ARHGAP12 kd, and ARHGAP29 kd cells with immunohisto- chemical staining (brown) and corresponding column graphs for expression of the following cell markers: (A) vimentin; (B) cleaved caspase-3 (CC3), an apoptosis marker; and (C) Ki67, expressed in proliferating cell nuclei. Black arrowheads indicate different morphological features of the tumor margin. Vimentin scale bar: 150 mm; CC3 and Ki67 scale bars: 75 mm. Data presented as mean ± SEM. (D and E) Both (D) N-cadherin and (E) E-cadherin were strongly expressed on tumor cells in the control group, with a significant loss of N-cadherin expression in ARHGAP12 kd tumors. Scale bar: 75 mm; post hoc Dunnett’s test. Data presented as median, interquartile range, and range.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Injection, Control, Staining, Expressing, Marker

Journal: Cell reports

Article Title: ARHGAP12 and ARHGAP29 exert distinct regulatory effects on switching between two cell morphological states through GSK-3 activity.

doi: 10.1016/j.celrep.2025.115361

Figure Lengend Snippet: Figure 7. The ARHGAPs regulate glioma cell migration via a novel GSK-3 signaling pathway Targeting GSK-3 activity with a small-molecule inhibitor (1) prevents b-catenin degradation by ubiquitination and (2) promotes b-catenin translocation to the nucleus, where it acts as a transcription factor. Transcription of ARHGAP12 and prevention of transcription of ARHGAP29 lead to changes in Src signaling and/or phosphorylation status of RhoA and Rac1 with (3) concomi- tant adoption of a less aggressive, ameboid phenotype in migratory cells. The phenotypic change in migrating cells may affect recurrence after surgery in patients.

Article Snippet: The following antibodies were used for immunocytochemistry studies and immunohistochemistry, Ki67 (1:5000, Abcam, Cambridge, UK; Cat # ab15580), Cleaved Caspase 3 (CC3) (1:100, Cell Signaling Technologies, New England, UK; Cat # D175), ARHGAP12 (1:200, Novus Biologicals, Cambridge, UK; Cat # NBP1-91678), ARHGAP29 (1:100, ATLAS Antibodies, Cambridge, UK; Cat # HPA026534), E-cadherin (1:100, Abcam, Cambridge, UK; Cat # ab1416), N-cadherin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany; Cat # Sc-59987), Vimentin (1:200, Abcam, Cambridge, UK; Cat # ab16700), Actin Cytoskeleton/Focal adhesion kit (1:500, Merck, Feltham, UK; Cat # FAK100).

Techniques: Migration, Activity Assay, Ubiquitin Proteomics, Translocation Assay, Phospho-proteomics

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 1 Characterization of ARHGAP29 knockdown keratinocytes. (A) Western blot for ARHGAP29 (A29, top panel) and Ponceau red staining of the same membrane (bottom; one representative of three experiments) of keratinocytes transduced with CRISPR scrambled (sc), CRISPR ARHGAP29 (#1, #2), shRNA scramble, shRNA ARHGAP29 (#2, #3), and sh#3 transduced with ARHGAP29 (+A29) or GFP (+GFP). (B) Quantification of ARHGAP29 protein levels. Values are the means (N = 3) ± SEM, **p < .01 and ***p < .001 following ordinary one-way ANOVA test with Tukey's multiple comparisons post-hoc test (only relevant comparisons are shown). (C) Representative phase contrast micrographs of scramble, CRISPR knockdown, shRNA knockdown (sh#3) and shRNA knockdown keratinocytes transduced with ARHGAP29-GFP (sh#3 + A29), all grown in KSFM. Scale bar = 100 μm. (D) Quantification of keratinocyte area under the conditions shown in C. Boxes display the 25–75th percentiles where the line represents the median and whiskers display the minimum to maximum values, *p < .05, ***p < .001 and ****p < .0001 following Kruskal–Wallis test with Dunn's multiple comparisons post-hoc test (only relevant comparisons are shown). N = 200–400 cells per group. ns, non-significant.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: Knockdown, Western Blot, Staining, Membrane, Transduction, CRISPR, shRNA

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 3 ARHGAP29 promotes keratinocyte proliferation. (A) Quantification of population doubling time. Values are the means ± SEM, *p < .05 following Brown–Forsythe test and Welch ANOVA test with Dunnett's T3 multiple comparisons post-hoc test. N = 6–8 per group. (B) Representative images of colony forming efficiency dishes with CRISPR scramble (CRISPRsc), CRISPR ARHGAP29 (CRISPR#1), shRNA scramble (shSc) and shRNA ARHGAP29 (sh#3) keratinocytes. (C) Quantification of colony area for all cell lines (including those represented in B). N = 752–1639 per group. Values are the means ± SEM, ****p < .0001 following Kruskal–Wallis test with Dunn's multiple comparisons post-hoc test (only comparisons to scrambled are shown). (D) Quantification of number of cells per colony for all cell lines (N = 9–12). Values are the means ± SEM, **p < .01, ****p < .0001 following ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test (only comparisons to scrambled are shown). (E) Quantification of number of cells per colony area (“cell density”) for all cell lines (N = 9–12). Values are the means ± SEM, *p < .05, ****p < .0001 following ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test. Only comparisons to scrambled are shown.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: CRISPR, shRNA

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 5 ARHGAP29 promotes collective cell migration. (A) Phase contrast micrographs of in vitro scratch wounds in confluent monolayers of shRNA scramble (shSc), shRNA ARHGAP29 (sh#3), and sh#3 transduced with ARHGAP29 (sh#3 + A29) keratinocytes grown in DMEM:HAM. Scale bar = 100 μm. T0 = 0 h after scratch and T 12 = 12 h after scratch. (B) Quantifications of the percentage of scratch closure over a 12-h period in all CRISPR and shRNA cell lines compared to their respective scrambled controls. Values are the means ± SEM, *p < .05 and **p < .01 after two-way ANOVA with Tukey's multiple comparisons post-hoc test. N = 6 per group.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: Migration, In Vitro, shRNA, Transduction, CRISPR

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

doi: 10.1002/dvdy.759

Figure Lengend Snippet: FIGURE 6 ARHGAP29 is present in embryonic, but not in adult keratinocytes, and is upregulated following wounding. (A)– (F) Immunofluorescent staining for ARHGAP29 (cyan) of murine wild-type skin of an unwounded E14.5 embryo (A), E18.5 embryo (B), adult (C), and of a 2-day (D), 4-day (E), and 7-day (F) wound. Nuclear DNA is stained with Hoechst (magenta). Scale bar = 50 μm; yellow arrow heads indicate the leading edge of the epidermis in open wounds and white dotted lines indicate the epidermal–dermal junction. HF, hair follicle. One representative image of N = 3 per time point. (G) Western blot analysis for ARHGAP29 and GAPDH protein levels of E14.5, E17.5 and adult unwounded skin extracts. (H) Quantification of ARHGAP29 levels shown in panel G normalized to GAPDH (loading) and to E14.5 values used as the reference developmental time point. Values are the means ± SEM, *p < .05 and after ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test. N = 3 per group. (I) Quantification of ARHGAP29 levels (using immunofluorescent signal) in keratinocytes at different time points during wound healing. Values are the means ± SEM, *p < .05 and **p < .01 after ordinary one-way ANOVA with Dunnett's multiple comparisons post-hoc test. N = 3 per group.

Article Snippet: The following antibodies were used for Western blotting: Rabbit polyclonal against ARHGAP29 (Novus Biologicals, Littleton, CO; catalog #NBP1-05989) was used at 1/1000; mouse monoclonal against GAPDH (Ambion Gibco Thermo Fisher Scientific; catalog AM#4300) was used at 1/9000; mouse anti-rabbit IgG-HRP (eBiosciences, San Diego, CA; catalog #18-8816-33) was used at 1/5000; sheep anti-mouse IgG–HRP (GE Healthcare, Chicago, IL; catalog #NA931V) was used at 1/5000.

Techniques: Staining, Western Blot