arhgap29  (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: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/eLife.85601

Figure Lengend Snippet: ( A ) RPE1 cells were transfected with siRNAs against ARHGAP29, caveolin-1, or non-targeting siRNAs and analysed by western blotting 48 hr post-transfection. ( B ) Quantification of western blot analysis shown in ( A ). Ratios normalised to the GAPDH loading control are displayed relative to the intensity of the control siRNA transfection for each protein indicated. Data represent the mean ± SD of 3–4 independent experiments. Statistical significance was calculated using an unpaired t -test. ns = p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001. ( C ) RPE1 cells were transfected with A2E-ARHGAP29 or NES-A2E, fixed, and stained with anti-Cav1 antibodies. Scale bar 5 µm. ( D ) Quantification of Cav1 rear localisation based on data shown in ( C ). Error bars indicate mean ± SEM *p≤0.05, ***p≤0.001, Wilcoxon test (n = 18 cells per condition). ( E ) Still images of RPE1 cells transfected with NES-A2E (top) or A2E-ARHGAP29 (bottom) imaged live by spinning disk confocal microscopy. Scale bars 10 µm. ( F–H ) Migration tracks ( F ), migration speed ( G ), and mean squared displacement ( H ) of RPE1 cells transfected with A2E-ARHGAP29 or NES-A2E. Quantification was performed on three independent experiments and a total of ~60 cells per sample. Statistical significance in ( G ) was calculated using an unpaired t -test; ***p≤0.001. Figure 6—source data 1. Original western blots shown in used for the quantification of data shown in . Figure 6—source data 2. Original pMLC western blots shown in used for the quantification of pMLC levels shown in .

Article Snippet: Antibody , Rabbit monoclonal anti-PARG1 (ARHGAP29) , Invitrogen , Cat# PA5-55336 RRID: AB_2645210 , WB: 1:1000.

Techniques: Transfection, Western Blot, Control, Staining, Confocal Microscopy, Migration

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/eLife.85601

Figure Lengend Snippet: ( A–D ) Migration tracks ( A ), migration speed ( B ), displacement ( C ), and mean squared displacement ( D ) of RPE1 cells transfected with esiRNAs against ARHGAP29 or non-targeting esiRNAs. Quantification was performed on two independent experiments. ( E ) RPE1 cells transfected with A2E-ARHGAP29 were fixed and stained with anti-Cav1 antibodies and fluorescently labelled phalloidin (Alexa Fluor 555). Scale bar 10 µm. Note that cortical areas enriched in ARHGAP29 show reduced Cav1 signals. The fluorescence intensities of ARHGAP29 and Cav1 in such cortical areas were measured and normalised against the intensities on the whole cell level. The ARHGAP29/Cav1 ratio is plotted, showing an approximately threefold enrichment of ARHGAP29 over Cav1. ( F ) RPE1 cells transfected with A2E-ARHGAP29 were fixed and stained with anti-PTRF/cavin1 antibodies and fluorescently labelled phalloidin (Alexa Fluor 555). Asterisks (*) indicate two untransfected control cells. White arrowheads indicate enrichment of PTRF/cavin1 at the cell rear. Red arrowheads indicate actin and ARHGAP29 at the cell rear. Scale bar 10 µm. ( G ) 2D and 3D heat maps of two untransfected control cells (asterisks in A ) and an A2E-ARHGAP29 transfected cells. White arrowheads indicate the position of the cell rear. Note the loss of PTRF/cavin1 enrichment at the cell rear in cells transfected with A2E-ARHGAP29.

Article Snippet: Antibody , Rabbit monoclonal anti-PARG1 (ARHGAP29) , Invitrogen , Cat# PA5-55336 RRID: AB_2645210 , WB: 1:1000.

Techniques: Migration, Transfection, Staining, Fluorescence, Control

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/eLife.85601

Figure Lengend Snippet: Low membrane tension promotes caveolae formation at the cell rear, whilst high membrane tension causes caveolae to flatten out, which is accompanied by the dissociation of cavin1/PTRF, EHD2, and Pacsin2 from membrane-embedded Cav1 scaffolds. The linkage between the cortical F-actin network and Cav1 scaffolds is also lost at high membrane tension. Caveolae or Cav1 scaffolds promote RhoA/ROCK signalling, MLC phosphorylation, and cell rear retraction, possibly by recruiting the RhoGEF Ect2 and ROCK to the cell rear. ARHGAP29 may be recruited to the cell rear at low membrane tension to suppress RhoA signalling, leading to reduced Cav1 Y14 phosphorylation, increased membrane tension, and caveolae flattening.

Article Snippet: Antibody , Rabbit monoclonal anti-PARG1 (ARHGAP29) , Invitrogen , Cat# PA5-55336 RRID: AB_2645210 , WB: 1:1000.

Techniques: Membrane

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/eLife.85601

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-PARG1 (ARHGAP29) , Invitrogen , Cat# PA5-55336 RRID: AB_2645210 , WB: 1:1000.

Techniques: Stable Transfection, Transfection, Construct, Recombinant, In Situ, Electron Microscopy, Sequencing, Modification, Protease Inhibitor, Magnetic Beads, Protein Quantitation, Transduction, esiRNA, Control, Clone Assay, Software