Journal: Frontiers in Genetics

Article Title: Targeted re-sequencing on 1p22 among non-syndromic orofacial clefts from Han Chinese population

doi: 10.3389/fgene.2022.947126

Figure Lengend Snippet: (A) Sanger sequencing results of the de novo harmful rare variant in ARHGAP29 . Sequence chromatograms indicate the heterozygous variant (NM_004815.3, NP_004806.3; c.1652G>C, p.R551T). The red letter and box emphasize the cross-species conservation of the altered amino acid. (B, C) Western blot and RT-qPCR analysis of the ARHGAP29 expression in HEK-293T cells 48 h after plasmid transfection. The results are presented as mean values with standard deviation (SD) normalized to GAPDH , and there were three biological replicates, *** p < 0.001.

Article Snippet: Subsequently, protein samples were separated by electrophoresis in agarose gels and transferred onto PVDF membranes, which were then blocked by 5% milk for 1 h and incubated with rabbit anti-human Arhgap29 antibody (Novus Biologicals, United States) at 4°C overnight, followed by incubation with anti-rabbit antibody (Proteintech, China) at room temperature for 1 h. At last, proteins were visualized by ECL substrate (Epizyme, China).

Techniques: Sequencing, Variant Assay, Western Blot, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Standard Deviation

Journal: Frontiers in Genetics

Article Title: Targeted re-sequencing on 1p22 among non-syndromic orofacial clefts from Han Chinese population

doi: 10.3389/fgene.2022.947126

Figure Lengend Snippet: Results of RNA sequencing on Arhgap29 R553T/R553T and wild-type mice. (A) Volcanic maps of differential expression genes. (B) GO analysis of DEGs. All the shown GO terms were significantly enriched with Q-value less than 0.05.

Article Snippet: Subsequently, protein samples were separated by electrophoresis in agarose gels and transferred onto PVDF membranes, which were then blocked by 5% milk for 1 h and incubated with rabbit anti-human Arhgap29 antibody (Novus Biologicals, United States) at 4°C overnight, followed by incubation with anti-rabbit antibody (Proteintech, China) at room temperature for 1 h. At last, proteins were visualized by ECL substrate (Epizyme, China).

Techniques: RNA Sequencing, Quantitative Proteomics

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: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Immunohistochemical staining of ArhGAP29-RhoA/ROCK1 in human endometrium. ArhGAP29 staining in normal human endometrium was more obvious compared with endometrial tissue with severe IUAs; whereas, immunohistochemical staining of RhoA/ROCK1 in human endometrium with severe IUAs was more obvious compared with normal endometrial tissue. Scale bar, 50 µm. IUAs, intrauterine adhesions; ArhGAP29, Rho GTPase activating protein 29; ROCK1, Rho-associated coiled-coil containing protein kinase 1.

Article Snippet: Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.

Techniques: Immunohistochemical staining, Staining

Journal: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Effect of miR-1291 antagomir on ArhGAP29-RhoA/ROCK1 pathway in murine uterine tissue. Immunofluorescence staining for ArhGAP29, RhoA and ROCK1 (green) and nuclear DNA (DAPI, blue) in murine uterine tissue from the IUAs group, miR-1291 antagomir group and the negative control group. Scale bar, 50 µm. ArhGAP29, Rho GTPase activating protein 29; ROCK1, Rho-associated coiled-coil containing protein kinase 1; IUAs, intrauterine adhesions; miR, microRNA.

Article Snippet: Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.

Techniques: Immunofluorescence, Staining, Negative Control

Journal: Molecular Medicine Reports

Article Title: MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model

doi: 10.3892/mmr.2017.7210

Figure Lengend Snippet: Effect of miR-1291 antagomir on ArhGAP29-RhoA/ROCK1 expression. (A) The expression levels of ArhGAP29, RhoA and ROCK1 were examined by reverse transcription-quantitative polymerase chain reaction. (B) Representative western-blot images and (C) densitometry of expression of ArhGAP29, RhoA and ROCK1 following western blotting analysis. ★ P<0.05, ★★ P<0.01 vs. the IUAs group; **P<0.01, ***P<0.001 vs. miR-1291 antagomir group. ArhGAP29, Rho GTPase activating protein 29; ROCK1, Rho-associated coiled-coil containing protein kinase 1; IUAs, intrauterine adhesions; miR, microRNA.

Article Snippet: Immunohistochemistry, using a commercial primary anti-ArhGAP29 antibody (NBP1-05989; Novus Biologicals Canada ULC, Oakville, ON, Canada), RhoA (sc179; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and ROCK1 (ab45171; 1:200; Abcam, Cambridge, MA, USA) were used to investigate the differential expression of ArhGAP29 and RhoA/ROCK1 in severe IUAs compared with normal endometrial tissue.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

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

Journal: JCI Insight

Article Title: Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling

doi: 10.1172/jci.insight.135385

Figure Lengend Snippet: ( A ) Candidate centrosomal ARHGAP proteins identified from cilia/centrosome databases and siRNA cilia screens. Six potential ARHGAPs were reported in at least 2 studies. ( B and C ) SiRNA knockdown in control cells (UCL93) demonstrated that reduced ARHGAP5, -29, and -35 expression resulted in a reduction of the percentage of ciliated cells and cilia length, whereas knockdown of ARHGAP1, -19, and -21 was neutral for ciliogenesis ( n = 3 independent experiments, N = 50 cells). ( D ) Centrosomal expression of endogenous ARHGAP35 in HEK293 cells was demonstrated using a proximity ligation assay with a myc-tagged BioID2-PACT fusion protein (BioID2-PACT), which localizes to centrosomes. In addition, endogenous ARHGAP35 was labeled by a second myc-tagged BioID2 fusion protein containing the C-terminus of PC1 (BioID2-CT1), indicating that both proteins are likely interaction partners. ( E and F ) There was reduced centrosomal expression of ARHGAP35 in PKD1 cystic (OX161) or null (c1, c2) cells compared with control (UCL93) cells in the proximity ligation assay using BioID-PACT ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Significance determined by 2-tailed Student’s t test ( F ). Significance determined by 1-way ANOVA corrected (Dunnett) for multiple comparison ( B and C) . PC1, polycystin-1.

Article Snippet: The following antibodies were used in this study: PC1 (7e12), PC2 (g20), actin, ARHGAP5, ARHGAP29, myc, streptavidin, GST (Santa Cruz Biotechnology), ArhGAP35, MLC and pMLC (Cell Signaling), Flag (Sigma), RhoA, Cdc42 and Rac1 (Cytoskeleton), and Arl13b (Proteintech).

Techniques: Knockdown, Control, Expressing, Proximity Ligation Assay, Labeling, Comparison