pak1 specific inhibitor ipa  (Tocris)

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 expression is adversely associated with the overall survival of ovarian cancer patients. (A) Genetic alterations of PAK1 across different cancer types, as retrieved from the cBioPortal database. (B) PAK1 expression patterns across a variety of cancers. (C) Association between PAK1 expression levels and copy number variations. (D) Correlation of PAK1 expression with homologous recombination (HR) status. (E) Kaplan–Meier survival analysis comparing ovarian cancer patient outcomes with low ( n = 467) and high ( n = 188) PAK1 expression levels.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Expressing, Homologous Recombination

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 regulates homologous recombination (HR) repair and olaparib sensitivity in ovarian cancer cells. (A, B) HR (A) and non-homologous end-joining (NHEJ) (B) repair efficiencies in control and PAK1-depleted HEK293T cells, evaluated using HR and NHEJ reporter systems. Data were presented as mean ± standard error of the mean from three independent experiments. (C) Western blotting analysis of PAK2 and PAK3 in control and PAK2/PAK3-depleted HEK293T cells. (D – G) HR (D, F) and NHEJ (E, G) repair efficiencies in control and PAK2/PAK3-depleted HEK293T cells, evaluated using HR and NHEJ reporter systems. (H, I) Cell cycle distribution in control and PAK1-depleted Ovcar8 cells, analyzed by flow cytometry. (J, K) RAD51 foci formation in Ovcar8 cells treated with 10 μM olaparib for 24 h: (J) representative images and (K) quantification. More than 200 cells were analyzed per experiment. (L, N) The survival of control or PAK1-depleted Ovcar8 (L) and SKOV-3 (N) cells, assessed by colony formation assay. (M, O) Phosphorylation of CHK1 in control or PAK1-depleted Ovcar8 (M) and SKOV-3 (O) cells, treated with 10 μM olaparib for 6 h. (P, Q) The survival of control, (P) PAK2-depleted, or (Q) PAK3-depleted Ovcar8 cells, assessed by colony formation assay. Error bars represent the standard error of the mean from three independent experiments. Scale bars = 50 μm.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Homologous Recombination, Non-Homologous End Joining, Control, Western Blot, Flow Cytometry, Colony Assay, Phospho-proteomics

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 regulates homologous recombination (HR) repair and olaparib sensitivity dependent on its kinase activity. PAK1-depleted cells were transfected with wild-type PAK1 or the K299R kinase mutant for 24 h. (A) Western blotting analysis of PAK1 and CHK1 phosphorylation in transfected Ovcar8 cells treated with 10 μM olaparib for 6 h. (B) The survival of transfected Ovcar8 cells treated with different concentrations of olaparib for 2 weeks, assessed by colony formation assay. (C) HR activity in transfected HEK293T cells co-transfected with HR reporter plasmids, followed by HR assay after 48 h. (D, E) RAD51 foci formation in transfected Ovcar8 cells treated with 10 μM olaparib for 24 h: (D) representative images and (E) quantification. Over 200 cells were analyzed in each experiment. Error bars represent the standard error of the mean from three independent experiments.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Homologous Recombination, Activity Assay, Transfection, Mutagenesis, Western Blot, Phospho-proteomics, Colony Assay

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 inhibition enhances the efficiency of olaparib in ovarian cancer cells. (A) Western blotting analysis of PAK1 and CHK1 phosphorylation in Ovcar8 cells treated with 10 μM olaparib, 10 μM IPA-3, or their combination for 6 h. (B) Homologous recombination (HR) efficiency in HEK293T cells transfected with HR reporter plasmids, treated with olaparib, IPA-3, or both, followed by HR assay. (C, D) RAD51 foci formation in Ovcar8 cells treated with olaparib, IPA-3, or their combination for 24 h: (C) representative images and (D) quantification. More than 200 cells were analyzed per experiment. (E, F) The survival of Ovcar8 (E) and SKOV-3 (F) cells treated with olaparib alone or in combination with IPA-3, assessed by colony formation assay. Error bars represent the standard error of the mean from three independent experiments. Statistical significance was determined by a two-tailed t -test, with P -values < 0.05 considered significant.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Inhibition, Western Blot, Phospho-proteomics, Homologous Recombination, Transfection, Colony Assay, Two Tailed Test

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 inhibition promotes olaparib-induced replication stress and DNA damage. (A, B) DNA fiber assay for the length of CIdU (red) tracks in Ovcar8 cells treated with olaparib, IPA-3, or their combination for 6 h: (A) representative images and (B) quantification. Data were expressed as mean ± standard deviation, analyzed by a two-tailed unpaired t -test. (C, D) Immunoblot analysis of chromatin and soluble fractions of Ovcar8 cells treated with olaparib, IPA-3, or both for 6 h, probing for the indicated antibodies (C), or IPOND (isolation of proteins on nascent DNA) analysis of RPA1 and RPA2 at replication forks (D). (E, F) γ-H2AX foci formation in Ovcar8 cells treated with olaparib, IPA-3, or their combination for 24 h: (E) representative images and (F) quantification. More than 100 cells were counted per experiment. (G) RNA sequencing analysis of Ovcar8 cells treated with olaparib and IPA-3. Differentially expressed genes were classified based on fold change ≥ 1.5 or ≤ 0.5, with P < 0.05. (H) Biological process analysis of up- and down-regulated genes in the combination treatment compared with IPA-3 alone. (I, J) GSEA of up- and down-regulated genes in the combination treatment compared with IPA-3 alone. (K) The heatmap displaying up-regulated genes associated with DNA repair. (L) The quantitative real-time PCR showed the up-regulated genes associated with DNA repair. Error bars represent the standard error of the mean from three independent experiments. Statistical significance was determined by a two-tailed t -test. Two-sided P -values < 0.05 were considered significant. Scale bars = 50 μm.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Inhibition, Standard Deviation, Two Tailed Test, Western Blot, Isolation, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: Combination of IPA-3 and olaparib synergistically suppresses ovarian cancer xenograft tumor growth. OVCAR8 and SKOV-3 cells were subcutaneously implanted into NOD-SCID mice, and the animals were treated with control (DMSO), IPA-3 (10 mg/kg), olaparib (50 mg/kg), or their combination (intraperitoneally, 3 days × 6 times). (A, B, L, M) Serum AST and ALT were measured for Ovcar8 (A, B) and SKOV-3 (L, M) xenografts. (C, D, N, O) Tumor images and growth curves for Ovcar8 (C, D) and SKOV-3 (N, O) xenografts. Data were expressed as mean ± standard error of the mean from five independent samples. Statistical significance was assessed by a two-tailed unpaired t -test. (E–K, P – V ) Hematoxylin-eosin, Ki-67, γ-H2AX, and cleaved caspase-3 staining in tumor tissues, evaluated by immunohistochemistry for Ovcar8 (E–K) and SKOV-3 (P–V) xenografts. Quantification is shown in the corresponding panels. Images of 10 random fields per section were analyzed using ImageJ software. Scale bars = 50 μm. Statistical analysis was performed using a two-tailed t -test and two-way ANOVA. P -values < 0.05 were considered significant.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Control, Two Tailed Test, Staining, Immunohistochemistry, Software

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: Combination of IPA-3 and olaparib synergistically suppresses ovarian cancer cells' growth in patient-derived organoid and patient-derived xenograft models. (A, B, D, E) Ovarian cancer organoids were treated with IPA-3 (200 nM), olaparib (200 nM), or both for 3 days. Representative bright-field images and quantitative analysis are shown. (C, F) Western blotting analysis of CHK1 phosphorylation and cleaved caspase-3 in ovarian cancer organoids treated with IPA-3 (200 nM), olaparib (200 nM), or both for 3 days. (G, H) Patient-derived xenograft models were established by transplanting tumor tissues into 6-week-old female BALB/c nude mice. Mice were treated with DMSO, IPA-3 (10 mg/kg), olaparib (50 mg/kg), or their combination. Tumor images (G) and growth curves (H) are shown. (I–O) Immunohistochemical analysis of hematoxylin-eosin, cleaved caspase-3, γ-H2AX, and Ki-67 levels in tumor tissues. Quantification of staining is shown in (K), (M), and (O). Data were represented as mean ± standard deviation. Images of 10 random fields per section were recorded for analysis. Statistical significance was assessed using a two-tailed t -test and a two-way ANOVA. P -values < 0.05 were considered significant. Scale bars = 50 μm.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Derivative Assay, Western Blot, Phospho-proteomics, Immunohistochemical staining, Staining, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

doi: 10.1038/s41467-024-47297-2

Figure Lengend Snippet: a c-kit + BM cells from diseased Gadd45g +/ − and Ctrl mice were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. b Representative immunofluorescence micrographs showing colocalization of GADD45g with RAC2 in c-kit + BM cells of Ctrl mice. Panels represent nucleus (blue), GADD45g (green), RAC2 (yellow), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 10 μm. c Representative immunofluorescence micrographs showing cellular distribution of GADD45g, RAC2 and RAC1 in cord blood CD34 + cells from healthy human donors. Panels represent nucleus (blue), GADD45g (green), RAC2 (orange), RAC1 (pink), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 5 μm. d Western blot analysis of RAC2-GTP and total RAC2 protein levels in c-kit + BM cells from diseased Gadd45g +/− and Ctrl mice. e HEL and SET-2 cells transfected with GADD45g-specific shRNA or shCtrl were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. f Western blot analysis of RAC2-GTP and total RAC2 protein levels in HEL and SET-2 cells transfected with GADD45g- specific shRNA or shCtrl. g Western blot analysis of p-PAK1 and total PAK1 protein levels in c-kit + BM cells from diseased Gadd45g +/ − and Ctrl mice. h Western blot analysis of p-PAK1 and total PAK1 protein levels in HEL and SET-2 cells transfected with GADD45g- specific shRNA or shCtrl. i HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by transfection with RAC2-specific shRNA (shRAC2) or scrambled control (Scr) for another 48 h. The protein levels of p-PAK1, total PAK1, p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. j HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by treatment with vehicle or IPA-3 (10 μM for 48 h). The protein levels of p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. For ( a–j ): At least three independent experiments with similar results were performed.

Article Snippet: AKT inhibitor MK-2206 2HCI, PAK1 inhibitor IPA-3, RAC inhibitor EHT 1864 2HCI, JAK2 inhibitor ruxolitinib, and HDAC1/2 inhibitor Romidepsin were all purchased from SelleckChem (Houston, Texas, USA).

Techniques: Western Blot, Immunofluorescence, Transfection, shRNA, Control

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: PI3K/AKT downstream Rac1-PAK1 signaling axis is not activated and involved in NDV entry into HD11 cells. (A) GTPase activity of Rac1 was analyzed by using the Rac1 activation assay kit, followed by Western blotting using an anti-Rac1 antibody. The level of GTP-Rac1 was normalized to the total Rac1. (B) The levels of p-PAK1 (T423) and PAK1 were analyzed by Western blotting using corresponding antibodies. GAPDH was used as a control. (C–G) Treatment with the Rac1 and PAK1 inhibitor, NSC23766 and IPA-3, showed no effect on NDV-induced LIMK1/CFN phosphorylation and entry of NDV. (C) HD11 cells were pretreated with NSC23766, IPA-3, or DMSO. Then the cells were inoculated or mock inoculated with NDV. Western blotting was used to determine the levels of p-LIMK1 (T508), LIMK1, p-CFN (S3), and CFN at 30 mpi with NDV. GAPDH was used as a control. (D–G) Treatment with the NSC23766 and IPA-3 had no effect on the adsorption and internalization of NDV. HD11 cells were pretreated with NSC23766 (D and E), IPA-3 (F and G), after which NDV adsorption and internalization assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Article Snippet: IPA-3 , PAK1 activation inhibitor , MCE , HY-15663.

Techniques: Activity Assay, Activation Assay, Western Blot, Control, Phospho-proteomics, Adsorption, Flow Cytometry

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Inhibitors and activators used in this study

Article Snippet: IPA-3 , PAK1 activation inhibitor , MCE , HY-15663.

Techniques: Activation Assay

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: IPA-3 , PAK1 activation inhibitor , MCE , HY-15663.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: High extracellular glucose promotes cell motility by modulating cell deformability and contractility via the cAMP-RhoA-ROCK axis in human breast cancer cells

doi: 10.1091/mbc.e22-12-0560

Figure Lengend Snippet: FIGURE 7: cAMP increases cell stiffness through activating RhoA-ROCK-NMII axis and increases actomyosin filament. (A) RhoA activity measured by GTP-RhoA pull-down assay with forskolin treatment. Quantitation of the total and active RhoA protein is normalized to vehicle treatment (below panel). Statistical significance was determined by unpaired t test (n = 3). (B) pMLC2 levels after forskolin treatment for 3 h with or without inhibitors for ROCK (Y27), MLCK (ML7), or PAK1 (IPA3). Quantitation is shown below (n = 3). (C) Representative confocal microscopy images of pMLC2 (green) and F-actin (red) from MDA-MB-231 cells treated with glucose or forskolin for 3 h. Scale bar, 10 μm. (D) Quantification of F-actin area per cell with increasing concentration of forskolin analyzed by Cellinsight CX7 HCA system (n = 3; 24 h). (E) Representative confocal microscopy images of F-actin from MDA-MB-231 cells treated with forskolin or cAMP analog for 24 h. Scale bar, 50 μm. (F) Total F-actin intensity per well measured from MDA-MB-231 cells treated with forskolin with or without Y27632 for 24 h (n = 8). (G) Subcellular localization of pMLC2 (green) and F-actin (red). Fluorescence intensity of each target in a marked region (white line) was analyzed using line intensity profiles (below each image). Pearson’s correlation coefficient representing colocalization efficiency is denoted. Scale bar, 10 μm (3 h). (H) Comparison of Pearson’s correlation coefficient (r) values from randomly selected marked

Article Snippet: Pharmacological inhibitors and activators were purchased from commercial vendors as following: Rho-associated kinase (ROCK) inhibitor, Y27632 (Selleck Chemicals, S6390), myosin light-chain kinase (MLCK) inhibitor, ML-7 (Selleck Chemicals, S8388), glycolysis inhibitor, 2-deoxy-D-glucose (2DG) (Sigma-Aldrich, D8375), p21activated kinase 1 (PAK1) inhibitor, IPA3 (Selleck Chemicals, S7093), Rho activator II, CN03 (Cytoskeleton, CN03), adenyl cyclase activator, Forskolin (Selleck Chemicals, S2449), and cAMP Analog, 8-Br-cAMP (Selleck Chemicals, S7857).

Techniques: Activity Assay, Pull Down Assay, Quantitation Assay, Confocal Microscopy, Concentration Assay, Fluorescence, Comparison

Journal: eLife

Article Title: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex

doi: 10.7554/eLife.67999

Figure Lengend Snippet: ( A ) Panels depict immunofluorescence of Yrt (green in merge) and Fasciclin 3 (Fas3; lateral marker, magenta) in the follicular epithelium treated for 2 hr with the aPKC inhibitor CRT-006-68-54. ( B ) aPKC knockdown cells (mRFP positive) were immunostained for Yrt. ( C ) crb 11A22 (null allele) homozygous mutant clones were produced in adult crb /+ female flies (mutant clones are GFP positive). Dissected ovaries were incubated with the aPKC inhibitor CRT-006-68-54 prior to fixation and Yrt immunostaining. ( D ) Quantification of apical Yrt intensity in control or crb mutant cells within the same follicle in presence of the aPKC inhibitor CRT-006-68-54. ( E ) Analysis of Yrt localization in control or crb 11A22 mutant cells expressing exogenous Crb Y10A (GFP-positive cells) exposed to the aPKC inhibitor CRT-006-68-54. ( F ) Quantification of apical Yrt staining in control and crb null cells expressing exogenous Crb Y10A treated with the aPKC inhibitor. ( G ) FLAG-Yrt 5A was specifically expressed in crb mutant cell clones (right panels). Mosaic expression of FLAG-Yrt 5A in control cells or crb mutant clones were used as controls (middle and left panels, respectively). Clones were labeled with GFP (left panels) or by Yrt immunostaining (middle and right panels). Dlg1 staining was used to label the lateral membrane. ( H ) Quantification of the apical domain width of cells expressing the transgenes listed in G. ( I ) Immunostaining of Yrt and Dlg1 in follicular epithelial cells expressing FLAG-Yrt 5A and exogenous wild-type Crb (left panels), or FLAG-Yrt 5A together with Crb Y10A (right panels). ( J ) Quantification of the apical domain width of cells expressing the transgenes listed in I. Results are expressed as the ratio between the width of cells expressing the transgenes and the width of control cells in the same follicle. Stage three follicles were depicted in A and B, whereas panels C , E , G , and I display stage five follicles. In D , F , H , and J error bars indicate s.d.; bold black lines denote the mean; n ≥ 20; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (one-way ANOVA). All scale bars represent 5 μm. Figure 5—source data 1. Crb cooperates with Yrt. Raw data for .

Article Snippet: Dissected ovaries were treated for 2 hr with inhibitors of aPKC (CRT-006-68-54, Bio-Techn Sales Corporation, 10 μM) or Pak1 (IPA-3, Millipore Sigma, 50 μM) as described ( ).

Techniques: Immunofluorescence, Marker, Knockdown, Mutagenesis, Clone Assay, Produced, Incubation, Immunostaining, Control, Expressing, Staining, Labeling, Membrane

Journal: eLife

Article Title: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex

doi: 10.7554/eLife.67999

Figure Lengend Snippet: ( A ) Mosaic knockdown of Moe in absence or presence of the aPKC inhibitor CRT-006-68-54. Clones are positively labeled with membrane-targeted RFP. ( B ) Quantification of apical domain width of knockdown cells in absence or presence of the aPKC inhibitor CRT-006-68-54. Results were calculated as a ratio between clonal cells and control cells in the same follicle. ( C ) Clonal expression of FLAG-Yrt, Myc-Moe T559D , or both proteins (clones are labeled with mRFP). ( D ) Quantification of the apical domain width of cells expressing the transgenes listed in C . Results are expressed as the ratio between the width of cells expressing transgenes and the width of control cells in the same follicle (quantification was performed using stage five follicles). In B and D , error bars indicate s.d.; bold black lines denote the mean; n ≥ 18; * p ≤ 0.05, **** p ≤ 0.0001 (one-way ANOVA). All scale bars represent 5 μm. Figure 6—source data 1. Moe and Yrt show antagonistic function. Raw data for .

Article Snippet: Dissected ovaries were treated for 2 hr with inhibitors of aPKC (CRT-006-68-54, Bio-Techn Sales Corporation, 10 μM) or Pak1 (IPA-3, Millipore Sigma, 50 μM) as described ( ).

Techniques: Knockdown, Clone Assay, Labeling, Membrane, Control, Expressing

Journal: eLife

Article Title: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex

doi: 10.7554/eLife.67999

Figure Lengend Snippet: ( A ) Yrt immunostaining in control follicular epithelial cells or clones of cells expressing aPKC CAAX together with Par6, Pak1 Myr , or aPKC CAAX with Par6 and Pak1 Myr . Clones were positively labeled with RFP or aPKC immunostaining in stage four follicles. Scale bar represents 5 μm. ( B ) Quantification of lateral Yrt staining intensity in genotypes described in A. Error bars indicate s.d.; bold black lines denote the mean; n ≥ 16; **** p ≤ 0.0001 (one-way ANOVA). ( C ) Control embryos, embryos overexpressing Par-6 and aPKC CAAX , or embryos expressing Pak1 Myr were homogenized. Samples were incubated or not with the λ phosphatase and processed for SDS-PAGE. Western blotting using anti-Yrt antibodies showed the migration profile of phosphorylated ( p Yrt) or unphosphorylated Yrt, whereas β-Tubulin (βTub) was used as loading control. ( D ) Western blots showing the migration profile of Yrt ( p Yrt stands for phosphorylated Yrt) extracted from wild-type (WT) or PP2A-A mutant embryos. The expression level of PP2A-A is also shown, and Actin was used as loading control. ( E ) Wild-type embryos were treated or not with the PP2A inhibitor Cantharidin, homogenized, and processed for SDS-PAGE. Western blotting using control samples shows the phosphorylation levels of Yrt ( p Yrt), whereas samples incubated with λ Phosphatase prior to gel electrophoresis show unphosphorylated Yrt. β-Tubulin (βTub) was used as loading control. ( F ) Embryos overexpressing Pak1 Myr were treated or not with PP2A inhibitor and homogenized. Western blotting using anti-Yrt antibodies showed the migration profile of phosphorylated ( p Yrt) or unphosphorylated Yrt. λ Phosphatase was used as a positive control of Yrt dephosphorylation, and β-Tubulin (βTub) was used as loading control. Figure 7—source data 1. aPKC and Pak1 control Yrt localization. Raw data for . Figure 7—source data 2. aPKC and Pak1 control Yrt phosphorylation. Original scan for . Figure 7—source data 3. PP2A dephosphorylates Yrt. Original scan for . Figure 7—source data 4. Pak1 acts upstream of PP2A. Original scan for . Figure 7—source data 5. Loading controls. Original scan of loading controls. Figure 7—source data 6. Annotated blots. Region of each blot shown in are highlighted with a yellow box.

Article Snippet: Dissected ovaries were treated for 2 hr with inhibitors of aPKC (CRT-006-68-54, Bio-Techn Sales Corporation, 10 μM) or Pak1 (IPA-3, Millipore Sigma, 50 μM) as described ( ).

Techniques: Immunostaining, Control, Clone Assay, Expressing, Labeling, Staining, Incubation, SDS Page, Western Blot, Migration, Mutagenesis, Phospho-proteomics, Nucleic Acid Electrophoresis, Positive Control, De-Phosphorylation Assay

Journal: eLife

Article Title: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex

doi: 10.7554/eLife.67999

Figure Lengend Snippet: Yrt is localized to the lateral membrane where it prevents the spread of apical characteristics in differentiating epithelial cells and controls the occluding function of septate junctions in fully differentiated cells ( ; ; ; ). Yrt also occupies the apical domain owing to its direct interaction with Crb . Yrt promotes Crb-dependent cortical tension (this study) in contrast to Moe that represses Myosin function downstream of Crb ( ; ). Hence, these proteins contribute to the functional plasticity of Crb, and a fine regulation of their association with Crb is thus required to define and stabilize the functional architecture of epithelial cells. Our data indicate that aPKC plays a key role in this process through phospho-dependent exclusion of Yrt from the apical domain. In contrast, Pak1 promotes Yrt dephosphorylation through activation of PP2A. The Pak1–PP2A module, which opposes aPKC function, is thus essential for Yrt-induced contractility. The equilibrium between aPKC and Pak1–PP2A activities thus balances Yrt and Crb functions.

Article Snippet: Dissected ovaries were treated for 2 hr with inhibitors of aPKC (CRT-006-68-54, Bio-Techn Sales Corporation, 10 μM) or Pak1 (IPA-3, Millipore Sigma, 50 μM) as described ( ).

Techniques: Membrane, Functional Assay, De-Phosphorylation Assay, Activation Assay

Journal: eLife

Article Title: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex

doi: 10.7554/eLife.67999

Figure Lengend Snippet: Detailed genotypes.

Article Snippet: Dissected ovaries were treated for 2 hr with inhibitors of aPKC (CRT-006-68-54, Bio-Techn Sales Corporation, 10 μM) or Pak1 (IPA-3, Millipore Sigma, 50 μM) as described ( ).

Techniques: