Review

rabbit anti pak2 (Cell Signaling Technology Inc)

Cell Signaling Technology Inc is a verified supplier

Cell Signaling Technology Inc manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Cell Signaling Technology Inc rabbit anti pak2
Rabbit Anti Pak2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pak2/PAK2+Antibody/10__1038_slash_s41467___026___78499___5-321-36-39
Average 95 stars, based on 212 article reviews

rabbit anti pak2 - by Bioz Stars, 2026-11

95/100 stars

Images

Control:

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium
Article Snippet: Protein concentration in clarified lysates was determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific) according to the manufacturer’s instructions, and equal amounts of total protein (20–30 μg per lane) were mixed with 4× Laemmli sample buffer, boiled for 5 min at 95°C, and resolved by SDS–PAGE. .. Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in TBST, and probed with the following primary antibodies: ARL13B (Cat# 17711-1-AP; Proteintech), PAK2 (Cat# 2608S; Cell Signaling Technology), PDGF-BB (Cat# NBP1-58279; Novus Biologicals), and β-actin (Cat# MA1-744; Thermo Fisher Scientific) as a loading control. .. HRP-conjugated anti-rabbit (Cat# 7074; Cell Signaling Technology) and anti-mouse (Cat# 7076; Cell Signaling Technology) secondary antibodies were used for chemiluminescence detection.

Cell-Signaling:

Article Title: Skeletal muscle Rac1 mediates exercise training adaptations towards muscle glycogen resynthesis and protein synthesis
Article Snippet: PAK1 , Cell Signaling Technology , #2602 , 1:1000 in 2 % skim milk. .. PAK2 , Cell Signaling Technology , #2608 , 1:1000 in 2 % skim milk. .. phospho(p)PAK1 thr(T)423/pPAK2 T402 , Cell Signaling Technology , #2601 , 1:1000 in 3 % BSA.

Electrophoresis:

Article Title: Integrated Mendelian Randomization and Single-Cell Transcriptomics Analysis Identifies Critical Blood Biomarkers and Potential Mechanisms in Epilepsy.
Article Snippet: The secondary antibodies used were Anti- Rabbit IgG (H + L) Secondary Antibody, HRP (#31460; 1:3000, Thermo Fisher), and Anti- Mouse IgG (H + L) Secondary Antibody, HRP (#13–4813- 85; 1:3000, Thermo Fisher). .. For protein capillary electrophoresis, the primary antibodies used were IQGAP1 (#ab133490, 1:50, Abcam), BKRB2 (#YN2508; 1:50, Immunoway), PAK2 (#2608S, 1:50, CST), ARPC1B (#28368- 1- AP, 1:50, Proteintech), and RAC2 (#10735- 1- AP; 1:50, Proteintech). .. The secondary antibodies used followed the manufacturer's instructions: Anti- Rabbit Secondary HRP Antibody (#042–206, 1:50, ProteinSimple), and Anti- Mouse Secondary HRP Antibody (#042–205, 1:50, ProteinSimple).

Article Title: Integrated Mendelian Randomization and Single‐Cell Transcriptomics Analysis Identifies Critical Blood Biomarkers and Potential Mechanisms in Epilepsy
Article Snippet: The secondary antibodies used were Anti‐Rabbit IgG (H + L) Secondary Antibody, HRP (#31460; 1:3000, Thermo Fisher), and Anti‐Mouse IgG (H + L) Secondary Antibody, HRP (#13–4813‐85; 1:3000, Thermo Fisher). .. For protein capillary electrophoresis, the primary antibodies used were IQGAP1 (#ab133490, 1:50, Abcam), BKRB2 (#YN2508; 1:50, Immunoway), PAK2 (#2608S, 1:50, CST), ARPC1B (#28368‐1‐AP, 1:50, Proteintech), and RAC2 (#10735‐1‐AP; 1:50, Proteintech). .. The secondary antibodies used followed the manufacturer's instructions: Anti‐Rabbit Secondary HRP Antibody (#042–206, 1:50, ProteinSimple), and Anti‐Mouse Secondary HRP Antibody (#042–205, 1:50, ProteinSimple).

other:

Article Title: Crystalline forms of carbazole inhibitors of RhoGTPase for the treatment of disease
Article Snippet: Four-parameter dose-response IC50 curves are fitted using the non-linear regression function of GraphPad Prism.

Image Search Results

A Gene set enrichment analysis on Fast-growing compared to parental cells ( n = 288 genes) and Endocrine Tolerant resistant cells compared to parental cells ( n = 288 genes) using Curated.MSigDB. B / C Steiner Forest signalling network analysis in upregulated genes ( n = 288) of B Fast-growing and C Endocrine Tolerant cells. Node size indicates number of attached edges, with nodes with >8 in bold. Node colours: Brown=genes, light blue=phenotype, dark blue=protein complex, pink=protein family. D Multiplexed analysis of EGFR phosphorylation in: basal cells, 30 min 100 ng/mL EGF, 30 min 100 nM 17 β-estradiol, or 30 min serum stimulation. Analysed by two-way ANOVA with Bonferroni’s multiple comparison test from n = 3 biological replicates, error bars=SD. E Western blot of EGFR, pEGFR (Y1045) and β-actin in Fast and Endocrine Tolerant cells after 100 ng/mL EGF treatment for indicated times, representative of 3 biological replicates. F Multiplexed analysis of HER2 phosphorylation following treatment described in D . Analysed by two-way ANOVA with Bonferroni’s multiple comparison test. G Western blot of HER2, pHER2 (Y1221/1222) and β-actin in Fast and Endocrine Tolerant cells after 100 ng/mL EGF stimulation for indicated times, representative of 3 biological replicates. (H) PREX1 transcripts from scRNAseq analysis of MCF-7 cells treated for 48 hours with fulvestrant ( n = 340 cells), and in Fast-growing ( n = 578 cells) and Endocrine Tolerant ( n = 659 cells). I Western blot of P-Rex1, Rac1, Pak2 and β-actin in Fast and Endocrine Tolerant cells, representative of 3 biological replicates. J Densitometry of P-Rex1 normalised to β-actin from n = 3 biological replicates. Analysed by two-sided t-test, error bars=SD. K Western blot of P-Rex1, ER and GAPDH in short-term fulvestrant and estrogen treated MCF-7 cells. Representative of 5 replicates. L Densitometry of P-Rex1 normalised to β-actin from n = 5 biological replicates, analysed by one-way ANOVA with Tukey’s multiple comparisons, error bars=SEM. M / N Multiplexed analysis of M pIGF1R and N pIR phosphorylation following treatment described in D . Analysed by two-way ANOVA with Bonferroni’s multiple comparisons. O Western blot of pIR/pIGF1R and β-actin in Fast and Endocrine Tolerant cells after 30 mins insulin. Representative of 3 replicates. P Western blot for P-Rex1 and GAPDH in Fast and Endocrine Tolerant cells expressing non-targeting (NT) and PREX1 shRNA, representative of n = 2 biological replicates. Q Quantitation of transwell migration assay of Fast and Endocrine Tolerant cells that express NT and PREX1 shRNA. Analysed by unpaired two-sided t-test from n = 3 biological replicates, error bars=SEM. R / S Correlation between PREX1 and EGFR in the (R) TCGA (RNAseq RSEM; n = 812 cancers) and S Metastatic Breast Cancer Project (RNAseq RSEM; n = 67 cancers). r=Spearman’s correlation. Orange line is least squares fit. Source data provided in Source Data file.

Journal: Nature Communications

Article Title: Endocrine therapy reprogramming of breast cancer facilitates metastatic escape via upregulation of P-Rex1/Rac1 signalling

doi: 10.1038/s41467-026-70683-x

Figure Lengend Snippet: A Gene set enrichment analysis on Fast-growing compared to parental cells ( n = 288 genes) and Endocrine Tolerant resistant cells compared to parental cells ( n = 288 genes) using Curated.MSigDB. B / C Steiner Forest signalling network analysis in upregulated genes ( n = 288) of B Fast-growing and C Endocrine Tolerant cells. Node size indicates number of attached edges, with nodes with >8 in bold. Node colours: Brown=genes, light blue=phenotype, dark blue=protein complex, pink=protein family. D Multiplexed analysis of EGFR phosphorylation in: basal cells, 30 min 100 ng/mL EGF, 30 min 100 nM 17 β-estradiol, or 30 min serum stimulation. Analysed by two-way ANOVA with Bonferroni’s multiple comparison test from n = 3 biological replicates, error bars=SD. E Western blot of EGFR, pEGFR (Y1045) and β-actin in Fast and Endocrine Tolerant cells after 100 ng/mL EGF treatment for indicated times, representative of 3 biological replicates. F Multiplexed analysis of HER2 phosphorylation following treatment described in D . Analysed by two-way ANOVA with Bonferroni’s multiple comparison test. G Western blot of HER2, pHER2 (Y1221/1222) and β-actin in Fast and Endocrine Tolerant cells after 100 ng/mL EGF stimulation for indicated times, representative of 3 biological replicates. (H) PREX1 transcripts from scRNAseq analysis of MCF-7 cells treated for 48 hours with fulvestrant ( n = 340 cells), and in Fast-growing ( n = 578 cells) and Endocrine Tolerant ( n = 659 cells). I Western blot of P-Rex1, Rac1, Pak2 and β-actin in Fast and Endocrine Tolerant cells, representative of 3 biological replicates. J Densitometry of P-Rex1 normalised to β-actin from n = 3 biological replicates. Analysed by two-sided t-test, error bars=SD. K Western blot of P-Rex1, ER and GAPDH in short-term fulvestrant and estrogen treated MCF-7 cells. Representative of 5 replicates. L Densitometry of P-Rex1 normalised to β-actin from n = 5 biological replicates, analysed by one-way ANOVA with Tukey’s multiple comparisons, error bars=SEM. M / N Multiplexed analysis of M pIGF1R and N pIR phosphorylation following treatment described in D . Analysed by two-way ANOVA with Bonferroni’s multiple comparisons. O Western blot of pIR/pIGF1R and β-actin in Fast and Endocrine Tolerant cells after 30 mins insulin. Representative of 3 replicates. P Western blot for P-Rex1 and GAPDH in Fast and Endocrine Tolerant cells expressing non-targeting (NT) and PREX1 shRNA, representative of n = 2 biological replicates. Q Quantitation of transwell migration assay of Fast and Endocrine Tolerant cells that express NT and PREX1 shRNA. Analysed by unpaired two-sided t-test from n = 3 biological replicates, error bars=SEM. R / S Correlation between PREX1 and EGFR in the (R) TCGA (RNAseq RSEM; n = 812 cancers) and S Metastatic Breast Cancer Project (RNAseq RSEM; n = 67 cancers). r=Spearman’s correlation. Orange line is least squares fit. Source data provided in Source Data file.

Article Snippet: Primary antibodies were cyclin A (1:1000, #sc-239), cyclin D1 (1:500, #sc-20044), estrogen receptor-α (human) (1:500, #sc-543), β-actin (1:15,000, #sc-69879), and GAPDH (1:15,000, #sc-32233) from Santa Cruz Biotechnology; p21 (1:1000, #610234), and total Rb (1:500, #554136) from BD Biosciences; P-Rex1 (1:1000, #HPA001927) from Sigma-Aldrich; Rac1 (1:1000, #ARC03) from Cytoskeleton Inc; PAK2 (1:2000, #A4553) from AB Clonal; estrogen receptor-α (mouse) (1:1000, #ab32063) from Abcam; EGFR (1:1000, #H00001956-M02) from Abnova; pHER2 (Tyr1221/1222) (1:1000, #2243); pEGFR (Tyr1045) (1:1000, #2237), HER2 (1:1000, #2165) and pERK (Thr202/Tyr204) (1:1000, #9101) from Cell Signalling Technology; and pIR/IGF1R (Tyr1162/Tyr1163) (1:1000, #44-804 G) from Invitrogen.

Techniques: Phospho-proteomics, Comparison, Western Blot, Expressing, shRNA, Quantitation Assay, Transwell Migration Assay, RNA sequencing

A Schematic of spontaneous tumour development and dissemination in the MMTV-PyMT model. Expression of Rac1 (A_51_P513254), Prex1 (A_51_P348372) and Pak2 (A_51_P172323) in stages of MMTV-PyMT development ( GSE43566 ) of adenoma ( n = 5), carcinoma ( n = 6), dissemination ( n = 6) and metastases ( n = 5). Data analysed by one-way ANOVA with Tukey’s multiple comparisons. B Schematic of derivation of MMTV-PyMT Rac1-FRET biosensor mice with biosensor in active (blue) and inactive (red) conformations, and derivation of a chronic tamoxifen biosensor cell line. C MMTV-PyMT Rac1-FRET parental (PyMT) and chronic tamoxifen (PyMT-Tam) cell lines western blotted for ER, P-Rex1, Pak2 and Rac1. β-actin loading control is shown for each western blot. Representative of three replicates. D Densitometry of P-Rex1 expression normalised to β-actin from triplicate biological replicates, analysed by two-sided t-test, and error bars represent SEM. E Representative image of Rac1-FRET activity in MMTV-PyMT cells chronically treated with tamoxifen with matched parental control. Scale bar = 100 µm. F Quantitation of Rac1-FRET activity of cells in E . Triplicate biological replicates analysed by two-sided t-test; error bars represent SEM. G Schematic of MMTV-PyMT Rac1-FRET biosensor mouse with titanium optical imaging window implanted over a mammary tumour. H Tumour growth of control ( n = 11) and tamoxifen ( n = 9) treated MMTV-PyMT Rac1-FRET mice. Data analysed by two-way ANOVA with a mixed effect model. Error bars represent SEM. I Image of Rac1-FRET activity in control and tamoxifen treated mammary tumours, representative of three biological replicates. Scale bar = 50 µm. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Endocrine therapy reprogramming of breast cancer facilitates metastatic escape via upregulation of P-Rex1/Rac1 signalling

doi: 10.1038/s41467-026-70683-x

Figure Lengend Snippet: A Schematic of spontaneous tumour development and dissemination in the MMTV-PyMT model. Expression of Rac1 (A_51_P513254), Prex1 (A_51_P348372) and Pak2 (A_51_P172323) in stages of MMTV-PyMT development ( GSE43566 ) of adenoma ( n = 5), carcinoma ( n = 6), dissemination ( n = 6) and metastases ( n = 5). Data analysed by one-way ANOVA with Tukey’s multiple comparisons. B Schematic of derivation of MMTV-PyMT Rac1-FRET biosensor mice with biosensor in active (blue) and inactive (red) conformations, and derivation of a chronic tamoxifen biosensor cell line. C MMTV-PyMT Rac1-FRET parental (PyMT) and chronic tamoxifen (PyMT-Tam) cell lines western blotted for ER, P-Rex1, Pak2 and Rac1. β-actin loading control is shown for each western blot. Representative of three replicates. D Densitometry of P-Rex1 expression normalised to β-actin from triplicate biological replicates, analysed by two-sided t-test, and error bars represent SEM. E Representative image of Rac1-FRET activity in MMTV-PyMT cells chronically treated with tamoxifen with matched parental control. Scale bar = 100 µm. F Quantitation of Rac1-FRET activity of cells in E . Triplicate biological replicates analysed by two-sided t-test; error bars represent SEM. G Schematic of MMTV-PyMT Rac1-FRET biosensor mouse with titanium optical imaging window implanted over a mammary tumour. H Tumour growth of control ( n = 11) and tamoxifen ( n = 9) treated MMTV-PyMT Rac1-FRET mice. Data analysed by two-way ANOVA with a mixed effect model. Error bars represent SEM. I Image of Rac1-FRET activity in control and tamoxifen treated mammary tumours, representative of three biological replicates. Scale bar = 50 µm. Source data are provided as a Source Data file.

Techniques: Expressing, Western Blot, Control, Activity Assay, Quantitation Assay, Optical Imaging

( a ) NISCH domain and its interacting partners. NISCH domains interacting with protein partners were drawn in lines in different colors. ( b-d ) Analysis of NISCH-protein interactions and Rac1 downstream pathway in MDA-MB-231 cells. MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG). ( b ) NISCH glutathionylation dissociates NISCH from Rac1 and PAK1. After incubating cells in LG or HG for 16 h, the co-immunoprecipitation was used to examine the binding of Rac1, PAK1, or integrin α5 (n = 2–3). ( c ) Rac1 activation analysis after NISCH C185 glutathionylation. After incubating cells for 16 h, the GST-fused p21-binding domain (PBD) derived from PAK1 (GST-PAK1-PBD) was used to pull down the Rac1-GTP form, which was then analyzed by Western blot (n = 2). ( d ) The co-localization analysis of NISCH and Rac1. After 16 h, cells were fixed and analyzed by antibodies to FLAG (green) and Rac1/Cdc42 (red) (n = 10 images). The merged images were analyzed to examine the relative displacement of NISCH and Rac1 in the cell periphery at higher magnification (box). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( b-d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Journal: Free radical biology & medicine

Article Title: Redox regulation of cell migration via Nischarin S-glutathionylation

doi: 10.1016/j.freeradbiomed.2025.11.013

Figure Lengend Snippet: ( a ) NISCH domain and its interacting partners. NISCH domains interacting with protein partners were drawn in lines in different colors. ( b-d ) Analysis of NISCH-protein interactions and Rac1 downstream pathway in MDA-MB-231 cells. MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG). ( b ) NISCH glutathionylation dissociates NISCH from Rac1 and PAK1. After incubating cells in LG or HG for 16 h, the co-immunoprecipitation was used to examine the binding of Rac1, PAK1, or integrin α5 (n = 2–3). ( c ) Rac1 activation analysis after NISCH C185 glutathionylation. After incubating cells for 16 h, the GST-fused p21-binding domain (PBD) derived from PAK1 (GST-PAK1-PBD) was used to pull down the Rac1-GTP form, which was then analyzed by Western blot (n = 2). ( d ) The co-localization analysis of NISCH and Rac1. After 16 h, cells were fixed and analyzed by antibodies to FLAG (green) and Rac1/Cdc42 (red) (n = 10 images). The merged images were analyzed to examine the relative displacement of NISCH and Rac1 in the cell periphery at higher magnification (box). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( b-d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: The membrane was blocked with 5 % BSA in TBST containing 50 mM Tris-HCl, 150 mM NaCl, and 0.1 % Tween-20, and incubated with primary antibody solution, containing phospho-PAK1 (p423) (1:1000; Cell Signaling, Cat# 2601S), PAK1 (1:1000; Cell Signaling, Cat# 2608S), phospho-cofilin-Ser-3 (Cell Signaling, Cat# 3311S), Cofilin (Cell Signaling, Cat# 3318S), phospho-LIMK-1 (Cell Signaling, Cat# 3841S), LIMK1 (Cell Signaling, Cat# 3842S), phosphor-paxillin (Cell Signaling, Cat# 441026G), Paxillin (Cell Signaling, Cat# 2542S) NISCH (D6T4X) (Cell Signaling, Cat# 85124S), phospho-p38 MAPK (1:1000; Cell Signaling, Cat# 9211S), p38 MAPK (1:1000; Cell Signaling, Cat# 9212S), phospho-MEK1/2 (1:1000; Cell Signaling, Cat# 9154S), MEK1/2 (1:1000; Cell Signaling, Cat# 9122S), phospho-p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling, Cat# 9101S), p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling, Cat# 9102S), phospho-JNK (1:1000; Cell Signaling, Cat# 9251S), JNK (1:1000; Cell Signaling, Cat# 9252S), phospho-p38 (1:1000; Cell Signaling, Cat# 9211S), p38 (1:1000; Cell Signaling, Cat# 9212S), Integrin α−5 (Cell Signaling, Cat# 4705S), GSTO1/2 (1:1000; Santa Cruz, Cat# sc-166040), GSTP (1:1000; MBL Life Science, Cat# 311-H), Glrx3 (1:1000; Sigma, Cat# SAB1410104), or FLAG antibody (1:1000; Millipore-Sigma, Cat# F3165), diluted in a blocking buffer and incubated for overnight at 4°C.

Techniques: Expressing, Incubation, Immunoprecipitation, Binding Assay, Activation Assay, Derivative Assay, Western Blot

MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG) for 16 h. ( a ) Phosphorylation levels of PAK1, ERK1/2, and MEK1/2 (n = 3). ( b ) Phosphorylation levels of LIMK1 and cofilin (n = 3). ( c ) Paxillin phosphorylation level (n = 3). ( d ) A model for cell migration induced by NISCH glutathionylation. In a non-stressed condition, NISCH binds to its protein partners, including Rac1 and PAK1, thus suppressing Rac1-and PAK1-mediated cell migration. NISCH may bind and retain Rac1 and integrin-α5 in the cytosol or endosome. However, upon ROS production, NISCH is glutathionylated at C185, dissociating Rac1 and PAK1, but not integrin-α5. The activated PAK1 activates its downstream LIMK1 and inhibits cofilin through phosphorylation, thereby increasing actin dynamics and polymerization. The dissociated Rac1 increases its localization to the membrane, where it activates actin filament branching, induces lamellipodia and membrane ruffles, and increases cell migration. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a – c ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Journal: Free radical biology & medicine

Article Title: Redox regulation of cell migration via Nischarin S-glutathionylation

doi: 10.1016/j.freeradbiomed.2025.11.013

Figure Lengend Snippet: MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG) for 16 h. ( a ) Phosphorylation levels of PAK1, ERK1/2, and MEK1/2 (n = 3). ( b ) Phosphorylation levels of LIMK1 and cofilin (n = 3). ( c ) Paxillin phosphorylation level (n = 3). ( d ) A model for cell migration induced by NISCH glutathionylation. In a non-stressed condition, NISCH binds to its protein partners, including Rac1 and PAK1, thus suppressing Rac1-and PAK1-mediated cell migration. NISCH may bind and retain Rac1 and integrin-α5 in the cytosol or endosome. However, upon ROS production, NISCH is glutathionylated at C185, dissociating Rac1 and PAK1, but not integrin-α5. The activated PAK1 activates its downstream LIMK1 and inhibits cofilin through phosphorylation, thereby increasing actin dynamics and polymerization. The dissociated Rac1 increases its localization to the membrane, where it activates actin filament branching, induces lamellipodia and membrane ruffles, and increases cell migration. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a – c ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Techniques: Expressing, Incubation, Phospho-proteomics, Migration, Membrane

(A, B) ELISAs performed on supernatants from control siRNA, PAK2 siRNA (A), and ARL3b siRNA (B) brain ECs with or without heparinase II treatment. (C, D, E) Immunofluorescent brain images of e11.5 WT Ext1 fl/fl mouse cortex stained for endomucin (green), PDGF-BB (red), and merged image. (F) Immunofluorescence image of PDGF-BB antibody stained (green) in a Tg( flk :mCherry) (red) 52-hpf zebrafish embryo. (F, F′, F″) White arrows point to PHBC. (F, F′, F″) Orange box in panel (F) is magnified in (F′, F″) showing PDGF-BB gradient-like expression close to red blood vessels in the brain. The scale bar in (F) is 100 μm.

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, B) ELISAs performed on supernatants from control siRNA, PAK2 siRNA (A), and ARL3b siRNA (B) brain ECs with or without heparinase II treatment. (C, D, E) Immunofluorescent brain images of e11.5 WT Ext1 fl/fl mouse cortex stained for endomucin (green), PDGF-BB (red), and merged image. (F) Immunofluorescence image of PDGF-BB antibody stained (green) in a Tg( flk :mCherry) (red) 52-hpf zebrafish embryo. (F, F′, F″) White arrows point to PHBC. (F, F′, F″) Orange box in panel (F) is magnified in (F′, F″) showing PDGF-BB gradient-like expression close to red blood vessels in the brain. The scale bar in (F) is 100 μm.

Article Snippet: Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in TBST, and probed with the following primary antibodies: ARL13B (Cat# 17711-1-AP; Proteintech), PAK2 (Cat# 2608S; Cell Signaling Technology), PDGF-BB (Cat# NBP1-58279; Novus Biologicals), and β-actin (Cat# MA1-744; Thermo Fisher Scientific) as a loading control.

Techniques: Control, Staining, Immunofluorescence, Expressing

(A, A′) IF for cilia ARL13b (green) and PDGF-BB (red) in brain microvascular ECs. (A, A′) Boxed region in panel (A) is zoomed in panel (A′). An arrow indicates a brain EC cilium that is colocalized with PDGF-BB. (A, A′) Scale bars: 20 μm for (A) and 10 μm for (A′). (B) Western blot of brain endothelial cell lysates from cells transfected with control or PDGF-BB siRNA. PDGF-BB, ARL13b, PAK2, and β-actin proteins were probed with specific antibodies and blots quantified by ImageJ. (B, C) Quantification data of the Western blots in panel (B). n = 3 in each experimental group. Results are presented as the mean ± SEM. Statistics were performed using paired t tests. P < 0.05 was considered significant. Source data are available for this figure.

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, A′) IF for cilia ARL13b (green) and PDGF-BB (red) in brain microvascular ECs. (A, A′) Boxed region in panel (A) is zoomed in panel (A′). An arrow indicates a brain EC cilium that is colocalized with PDGF-BB. (A, A′) Scale bars: 20 μm for (A) and 10 μm for (A′). (B) Western blot of brain endothelial cell lysates from cells transfected with control or PDGF-BB siRNA. PDGF-BB, ARL13b, PAK2, and β-actin proteins were probed with specific antibodies and blots quantified by ImageJ. (B, C) Quantification data of the Western blots in panel (B). n = 3 in each experimental group. Results are presented as the mean ± SEM. Statistics were performed using paired t tests. P < 0.05 was considered significant. Source data are available for this figure.

Techniques: Western Blot, Transfection, Control

(A, B) qRT-PCR for indicated HS proteoglycan gene synthesis and modifying enzyme targets in PAK2 siRNA ECs (A) and ARL13b siRNA ECs (B), respectively. (C) HS disaccharide analysis in control and PAK2 siRNA brain ECs. (D) PDGF-BB cell surface ELISA binding assay on control, PAK2 siRNA brain ECs, and ARL13b siRNA brain ECs. Heparinase treatment is used as a positive control. (E, F) Panels are immunofluorescent HSPG antibody stained (green) in pak2a WT (E) or pak2a homozygous bleeder (F) 52-hpf embryo in a Tg( flk :mCherry) (red) fish background. MCeV stands for mid-cerebral vein. The arrow points to region where HSPGs are stained in MCeV, which is surrounding the blood vessel outline. Scale bars in (E, F) are 100 μm. (A, B) T test was used for statistical analysis of qRT-PCR data in (A, B).

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, B) qRT-PCR for indicated HS proteoglycan gene synthesis and modifying enzyme targets in PAK2 siRNA ECs (A) and ARL13b siRNA ECs (B), respectively. (C) HS disaccharide analysis in control and PAK2 siRNA brain ECs. (D) PDGF-BB cell surface ELISA binding assay on control, PAK2 siRNA brain ECs, and ARL13b siRNA brain ECs. Heparinase treatment is used as a positive control. (E, F) Panels are immunofluorescent HSPG antibody stained (green) in pak2a WT (E) or pak2a homozygous bleeder (F) 52-hpf embryo in a Tg( flk :mCherry) (red) fish background. MCeV stands for mid-cerebral vein. The arrow points to region where HSPGs are stained in MCeV, which is surrounding the blood vessel outline. Scale bars in (E, F) are 100 μm. (A, B) T test was used for statistical analysis of qRT-PCR data in (A, B).

Techniques: Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Staining

(A, B) Quantification of EC ARL13b cilium numbers (A) and ARL13b cilium length (B) in control, PAK2 siRNA brain ECs, and ARL13b siRNA brain ECs with and without heparinase (H) treatment. (C) Panel shows quantification of the number of EC ARL13b cilia in n = 8 pak2a homozygous bleeders (B) (mi149/mi149), n = 6 pak2a heterozygous non-bleeders (NB) (mi149/WT), and n = 5 pak2a WT (WT/WT). (D, E) Whole-mount images of control vehicle (D) or human PDGF-BB protein (1 ng)–injected zebrafish 52-hpf embryo. (D, E) Note brain bleeding is reduced in panel (E) compared with panel (D). (D, E, F) Quantification of the bleeding area in panels (D, E). N = 6 for control vehicle–injected and N = 5 for PDGFB-BB protein–injected embryos. Scale bars in (D) are 200 μm. (C) GLM with negative binomial distribution was performed to compare the number of cilia among group in (C). Dunnett’s test was used to adjust for multiple comparisons. Estimated means and 95% confidence intervals (CIs) were used for plotting the number of cilia. WT had more cilia compared with group B or NB, adjusted P = 0.0018 and 0.013, respectively. (F) Unpaired t test was used to analyze bleeding data in (F). ** P = 0.0031. (G, H) Immunofluorescent ARL13b antibody–stained (green) images of human PDGF-BB protein (1 ng)–injected (G) or control vehicle–injected (H) Tg( flk :mCherry) rhd mi149/mi149 (homozygous) bleeders. White arrows in (G, H) show Keyence microscope measurement of dimensions of the area analyzed for quantitation of endothelial cilia: 160 μm posterior to the MTA/MCeV and 90 μm dorsal to PHBC. MTA, metencephalic artery; MCeV, middle cerebral vein; PHBC, primordial hindbrain channels. (G, G′, H, H′) White boxes in panels (G, H) are magnified in panels (G′, H′). (G′, H′) Note: Many cilia are seen in panel (G′) (white arrows) compared with panel (H′). Panel (I) is the quantification of the number of endothelial cilia in the human PDGF-BB protein (1 ng)–injected and control vehicle–injected rhd mi149/mi149 (homozygous) bleeders. N = 3 embryos were quantified in each group. Scale bars in (G, G′, H, H′) are 50 μm. An unpaired t test was used to analyze differences in the number of cilia, which were found to be not statistically significant.

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, B) Quantification of EC ARL13b cilium numbers (A) and ARL13b cilium length (B) in control, PAK2 siRNA brain ECs, and ARL13b siRNA brain ECs with and without heparinase (H) treatment. (C) Panel shows quantification of the number of EC ARL13b cilia in n = 8 pak2a homozygous bleeders (B) (mi149/mi149), n = 6 pak2a heterozygous non-bleeders (NB) (mi149/WT), and n = 5 pak2a WT (WT/WT). (D, E) Whole-mount images of control vehicle (D) or human PDGF-BB protein (1 ng)–injected zebrafish 52-hpf embryo. (D, E) Note brain bleeding is reduced in panel (E) compared with panel (D). (D, E, F) Quantification of the bleeding area in panels (D, E). N = 6 for control vehicle–injected and N = 5 for PDGFB-BB protein–injected embryos. Scale bars in (D) are 200 μm. (C) GLM with negative binomial distribution was performed to compare the number of cilia among group in (C). Dunnett’s test was used to adjust for multiple comparisons. Estimated means and 95% confidence intervals (CIs) were used for plotting the number of cilia. WT had more cilia compared with group B or NB, adjusted P = 0.0018 and 0.013, respectively. (F) Unpaired t test was used to analyze bleeding data in (F). ** P = 0.0031. (G, H) Immunofluorescent ARL13b antibody–stained (green) images of human PDGF-BB protein (1 ng)–injected (G) or control vehicle–injected (H) Tg( flk :mCherry) rhd mi149/mi149 (homozygous) bleeders. White arrows in (G, H) show Keyence microscope measurement of dimensions of the area analyzed for quantitation of endothelial cilia: 160 μm posterior to the MTA/MCeV and 90 μm dorsal to PHBC. MTA, metencephalic artery; MCeV, middle cerebral vein; PHBC, primordial hindbrain channels. (G, G′, H, H′) White boxes in panels (G, H) are magnified in panels (G′, H′). (G′, H′) Note: Many cilia are seen in panel (G′) (white arrows) compared with panel (H′). Panel (I) is the quantification of the number of endothelial cilia in the human PDGF-BB protein (1 ng)–injected and control vehicle–injected rhd mi149/mi149 (homozygous) bleeders. N = 3 embryos were quantified in each group. Scale bars in (G, G′, H, H′) are 50 μm. An unpaired t test was used to analyze differences in the number of cilia, which were found to be not statistically significant.

Techniques: Control, Injection, Staining, Microscopy, Quantitation Assay

Review

rabbit anti pak2 (Cell Signaling Technology Inc)

Structured Review

Images

Related Articles

Control:

Cell-Signaling:

Electrophoresis:

other:

Similar Products

Image Search Results