gtpase 13b  (Proteintech)

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, C) pak2a +/+ WT, pak2a rhd mi149/+ (heterozygous) non-bleeder (NB), and pak2a rhd mi149/mi149 (homozygous) bleeder (B), respectively, at 52-h post-fertilization (hpf) transgenic zebrafish embryos derived from an incross of pak2a rhd mi149/+ heterozygous in the background of zebrafish line Tg( pdgfrb : GFP; kdrl : mCherry; and beta-actin : Arl13b-GFP). (A′, B′, C′) Magnified regions of the white dotted boxes in the respective panels. (A′, B′, C′) Star-shaped cells ((B′, C′), red asterisk) are long-arm pericytes and short-arm pericytes ((A′, B′, C′), white asterisk), and white arrows (B′) are cilia. (D) Number of pdgfrb + pericytes quantified between rhd WT, heterozygous (mi149/WT), and homozygous (mi149/mi149) embryos. (A′, B′, C′) Scale bars in (A, B, C) are 100 μm, and in (A′, B′, C′) are 30 μm. A rectangular area (length = 190 μm posterior to the MCeV and width = 150 μm dorsal to the PHBC) was analyzed in all images. (E) Distance of pericyte from endothelial cilia. (F, G, H) Distance of pericytes from vessels (F), and pericyte distribution from vessels with (G) and without (H) cilia are plotted across the three sample groups. (F, G, H) Panels are box plots. * P < 0.05. n = 8 pak2a homozygous bleeders (B) (mi149/mi149), n = 6 pak2a heterozygous non-bleeders (NB) (mi149/WT), and n = 5 pak2a WT (WT/WT). Panel (F) shows estimated means and 95% confidence intervals (CIs). Panel (G) shows means and standard error of means. Results were not statistically significant. (D, E) GLM with the Poisson distribution or ANOVA with Dunnett’s test for multiple comparisons adjustment was used for statistical analysis of panels (D, E), respectively, and results were not statistically significant. (E, F) Adjusted P = 0.37 (WT versus B) and P = 0.27 (WT versus NB); (F) and P = 0.95 (WT versus B) and P = 0.88 (WT versus NB) (E). Panel (F) shows all data points and box plots. The Kruskal–Wallis test was used. P = 0.68. For panels (G, H), all data points and box plots were plotted. (G, H) Kruskal–Wallis test along with Dwass–Steel–Critchlow–Fligner method for multiple comparisons adjustment (G, H) was used for statistical analysis. Bleeders versus WT groups were compared and P = 0.04 (G) and P = 0.04 (H).

Article Snippet: For PDGF-BB:cilium rescue experiments , we performed immunofluorescence experiment using ARL13B primary antibody (Cat# 17711-1-AP, dilution 1:50; Proteintech) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) secondary antibody (green) (Cat# A-21206,1:250; Thermo Fisher Scientific).

Techniques: Transgenic Assay, Derivative Assay

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) Human microvascular brain ECs cultured in a microfluidic chamber and stained for ciliary marker ARL13b and acetylated α-tubulin in the cross-sectional plane; the white dotted box was enlarged in the middle. (B) Transverse plane of the vessel tube. White arrowheads indicate ARL13b cilia projecting into the basolateral space. Scale bars: 100 μm. (C, C′) Fluorescent image of a 52-hpf transgenic zebrafish Tg( flk :mCherry; b-actin :Arl13b-GFP) expressing mCherry in the vasculature and mouse Arl13b-GFP fusion protein in the cilia. (C′) White boxed area is enlarged in (C′). White arrows in (C′) point to basolateral zebrafish endothelial cilium location. The scale bar in (C, C′) is 50 μm. (D) Quantification of zebrafish endothelial cilia (luminal versus abluminal location) in metencephalic artery (MTA) and middle cerebral vein (MCeV). N = 4 for each vessel group was quantified for the presence of abluminal versus luminal cilia. An unpaired t test showed no difference in the cilium location between the two vessel groups. (E) Immunohistochemistry for abluminal cilia from representative cortical microvessels from ARL13b-EGFP transgenic adult mice ( C57BL/6 background) showing luminal versus abluminal cilium localization stained with anti-GFP. The black arrow points to Arl13b-positive abluminal cilia. Scale bars: 100 μm. (E′) Quantification of Arl13b cilium length on the abluminal versus luminal side of mouse endothelial vessels. (E″) Frequency of cilia on the abluminal versus luminal side of mouse endothelial vessels. A total of 30 endothelial vessel-like structures were quantified. (F) Immunofluorescence staining of a 22-wk-old human brain for ARL13b protein (green) and CD31 vessel staining (red). White arrows point to cilia in the basolateral side of the vessel. Scale bars: 10 μm. (F′) Quantification of endothelial cilial frequency in the ventricular zone (VZ) and subventricular zone (SVZ) in human brain sections. Scale bar in (F) is 10 μm.

Article Snippet: For PDGF-BB:cilium rescue experiments , we performed immunofluorescence experiment using ARL13B primary antibody (Cat# 17711-1-AP, dilution 1:50; Proteintech) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) secondary antibody (green) (Cat# A-21206,1:250; Thermo Fisher Scientific).

Techniques: Cell Culture, Staining, Marker, Transgenic Assay, Expressing, Immunohistochemistry, Immunofluorescence

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.

Article Snippet: For PDGF-BB:cilium rescue experiments , we performed immunofluorescence experiment using ARL13B primary antibody (Cat# 17711-1-AP, dilution 1:50; Proteintech) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) secondary antibody (green) (Cat# A-21206,1:250; Thermo Fisher Scientific).

Techniques: Western Blot, Transfection, Control

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).

Article Snippet: For PDGF-BB:cilium rescue experiments , we performed immunofluorescence experiment using ARL13B primary antibody (Cat# 17711-1-AP, dilution 1:50; Proteintech) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) secondary antibody (green) (Cat# A-21206,1:250; Thermo Fisher Scientific).

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

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.

Article Snippet: For PDGF-BB:cilium rescue experiments , we performed immunofluorescence experiment using ARL13B primary antibody (Cat# 17711-1-AP, dilution 1:50; Proteintech) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) secondary antibody (green) (Cat# A-21206,1:250; Thermo Fisher Scientific).

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