β1 integrin goat pab  (R&D Systems)

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 8. β1-integrin is an SNX17 cargo in neurons and plays a role in dendritic spine density. (A) DIV11 rat cortical neurons were infected with len- tiviruses carrying scrambled or SNX17 shRNAs, and the surface levels of β1-integrin were determined at DIV17 using a surface biotinylation assay. SNX17 knockdown was validated by Western blot of the lysate and GAPDH was used as a loading control. (B) The levels of surface β1-integrin protein were quantified and normalized to total β1-integrin levels (lysate). Data are expressed as a percentage of ctrl-shRNA (ctrl-shRNA: 100%, SNX17-shRNA: 57.630 ± 3.058%). N = 4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. (C) Rep- resentative confocal images of surface β1-integrin levels of DIV17 hippocampal neurons that were infected at DIV11 with lentiviruses carrying either ctrl-shRNA or SNX17-shRNA. Neurons were treated in the presence or absence of cLTP and live labeled with an anti-surface ß1-integrin antibody for 15 min, followed by fixation and immunostaining for MAP2. Scale bar, 5 µm. (D) The intensity of ß1-integrin in the first 50 µm of secondary dendrites was quantified and values were normalized to crtl-shRNA. ctrl-shRNA: 1.000 ± 0.038, N = 32 neurons; ctrl-shRNA cLTP: 1.139 ± 0.039, N = 29 neurons; SNX17-shRNA: 0.839 ± 0.040, N = 28 neurons; SNX17-shRNA cLTP: 0.786 ± 0.035, N = 28 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, *P < 0.05. Error bars are SEM. (E) Validation of an shRNA clone (V2LMM_39157, Horizon Discovery) to knockdown rat ITGB1. pGIPZ scrambled non- target (RHS4346, Horizon Discovery) was used as a control. HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or ITGB1-shRNA in the absence or presence of eGFP or ITGB1-GFP, as indicated. 5 d post-infection, cells were treated with 1 μg/ml of dox- ycycline to promote the expression of eGFP or ITGB1-GFP. 24 h later, extracts were generated and analyzed by Western blot. (F) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or ITGB1-shRNA. Scale bar, 5 µm. Treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation. Scale bar, 5 µm. (G) The numbers of dendritic spines in the first 30 μm of secondary dendrites were quantified. ctrl-shRNA: 0.705 ± 0.044, N = 31 neurons; ITGB1-shRNA: 0.505 ± 0.043, N = 33 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. (H) Hippocampal neurons were transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or SNX17-shRNA. Neurons were treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation at DIV16. The number of dendritic spines in the first 30 μm of secondary dendrites was quantified. ctrl-shRNA + isotype ctrl: 0.689 ± 0.030, N = 26 neurons; ctrl-

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Infection, Surface Biotinylation Assay, Knockdown, Western Blot, Control, shRNA, Two Tailed Test, Labeling, Immunostaining, Biomarker Discovery, Stable Transfection, Expressing, Transfection, Generated, Blocking Assay

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 9. β1-integrin has roles in structural and functional plasticity during cLTP. (A) Diagram of experiment. DIV16-18 hippocampal neurons were treated with either β1-integrin blocking or isotype control antibodies for 30 min, followed by a 5 min cLTP stimulus. mEPSCs were recorded during the first 30 min after cLTP (cLTP < 30) or from 30 to 90 min after cLTP (cLTP > 30). (B) Examples of mEPSC recordings of neurons that were treated with isotype ctrl or β1-integrin blocking antibodies. Recordings were performed in the absence of cLTP (baseline), during the first 30 min after cLTP, or from 30 to 90 min after cLTP. (C) Quantification of mEPSC amplitude. Isotype ctrl baseline: 12.240 ± 0.570, N = 19 neurons; isotype ctrl cLTP < 30 min: 16.790 ± 1.106, N = 9 neurons; isotype ctrl cLTP > 30 min: 16.900 ± 0.954, N = 10 neurons; β1-integrin blocking baseline: 11.570 ± 0.373, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 18.110 ± 1.247, N = 6 neurons; β1-integrin blocking neurons cLTP > 30 min: 13.170 ± 0.458, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) Quantification of mEPSC frequency. Isotype ctrl baseline: 1.191 ± 0.227, N = 19 neurons; isotype ctrl cLTP < 30 min: 3.333 ± 0.800, N = 9 neurons; isotype ctrl cLTP > 30 min: 3.110 ± 0.740, N = 10 neurons; β1-integrin blocking baseline: 1.167 ± 0.296, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 3.952 ± 1.214, N = 6 neurons; β1-integrin blocking cLTP > 30 min: 1.096 ± 0.302, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. (E) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. Neurons were treated with β1- integrin blocking or isotype control antibodies for 30 min, followed by a 5-min cLTP stimulus in the presence of antibodies where indicated. Neurons were further incubated in the presence of antibodies for 50 min before fixation. Scale bar, 5 µm. (F) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. Isotype ctrl: 0.626 ± 0.016, N = 28 neurons; isotype ctrl with cLTP: 0.717 ± 0.018, N = 25 neurons; β1-integrin blocking: 0.621 ± 0.015, N = 31 neurons; β1-integrin blocking with cLTP: 0.628 ± 0.010, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***P < 0.005. Error bars are SEM. (G) Quantification of dendritic spine density (spines/μm). Isotype ctrl: 1.044 ± 0.048, N = 28 neurons; isotype ctrl with cLTP: 0.977 ± 0.050, N = 25 neurons; β1-integrin blocking: 0.931 ± 0.054, N = 31 neurons; β1-integrin blocking with cLTP: 0.931 ± 0.039, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Functional Assay, Blocking Assay, Control, Transfection, Incubation

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 10. Model of SNX17-mediated modulation of synaptic structure and function. The SNX17-Retriever pathway is required for dendritic spine maintenance and the cLTP-dependent increase in dendritic spine size. Glycine-mediated cLTP (1) stimulates calcium entry through the NMDA receptor, which activates the CaMKII pathway (2). CaMKII activation is necessary and sufficient to promote the recruitment of SNX17 and the Retriever complex to dendritic spines (3), and activates the recycling of β1-integrin from endosomes to the plasma membrane (4). The surface levels of β1-integrin increase during cLTP and promote dendritic spine growth (5). Endosomal PI(3)P increases upon cLTP and may help with the recruitment of SNX17 to synapses. Created with BioRender. com.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Activation Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity

doi: 10.1101/2023.02.20.529299

Figure Lengend Snippet: β1-integrin is a SNX17 cargo in neurons and plays a role in dendritic spine density. (A) DIV11 rat cortical neurons were infected with lentiviruses carrying scramble or SNX17 shRNAs, and the surface levels of β1-integrin were determined at DIV17 using a surface biotinylation assay. SNX17 knockdown was validated by western blotting of the lysate, and GAPDH was used as a loading control. (B) The levels of surface β1-integrin protein were quantified and normalized to total β1-integrin levels (lysate). Data are expressed as percentage of ctrl-shRNA (ctrl-shRNA: 100%, SNX17-shRNA: 57.630 ± 3.058%). N=4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t-test, ****p<0.001. Error bars are SEM. (C) Representative confocal images of surface β1-integrin levels of DIV17 hippocampal neurons that were infected at DIV11 with lentiviruses carrying either ctrl-shRNA or SNX17-shRNA. Neurons were treated in the presence or absence of cLTP and live labeled with an anti-surface ß1-integrin antibody for 15 min, followed by fixation and immunostaining for MAP2. Scale bar, 5 µm. (D) The intensity of ß1-integrin in the first 50 µm of secondary dendrites was quantified, and values were normalized to crtl-shRNA. ctrl-shRNA: 1.000 ± 0.038, N=32 neurons; ctrl-shRNA cLTP: 1.139 ± 0.039, N=29 neurons; SNX17-shRNA: 0.839 ± 0.040, N=28 neurons; SNX17-shRNA cLTP: 0.786 ± 0.035, N=28 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, *p<0.05. Error bars are SEM. (E) Validation of a shRNA clone (V2LMM_39157, Horizon Discovery) to knock-down rat ITGB1. pGIPZ scrambled non-target (RHS4346, Horizon Discovery) was used as a control. HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or ITGB1-shRNA in the absence or presence of eGFP or ITGB1-GFP, as indicated. 5-days post-infection, cells were treated with 1 μg/ml of doxycycline to promote the expression of eGFP or ITGB1-GFP. 24 hours later, extracts were generated and analyzed by western blot. (F) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or ITGB1-shRNA. Scale bar, 5 µm. treated with either β1-integrin blocking or iso type control antibodies 24 hours before fixation. Scale bar, 5 µm. (G) The numbers of dendritic spines in the first 30 μm of secondary dendrites were quantified. ctrl-shRNA: 0.705 ± 0.044, N=31 neurons; ITGB1-shRNA: 0.505 ± 0.043, N=33 neurons. Statistical significance was determined using unpaired two-tailed Student’s t-test, **p<0.01. Error bars are SEM. (H) Hippocampal neurons were transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or SNX17-shRNA. Neurons were treated with either β1-integrin blocking or isotype control antibodies 24 hours before fixation at DIV16. The number of dendritic spines in the first 30 μm of secondary dendrites was quantified. ctrl-shRNA + isotype ctrl: 0.689 ± 0.030, N=26 neurons; ctrl-shRNA +β1-integrin blocking: 0.451 ± 0.021, N=27 neurons; SNX17-shRNA + isotype ctrl: 0.385 ± 0.026, N=28 neurons; SNX17-shRNA +β1-integrin blocking: 0.387 ± 0.020, N=26 neurons,. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****p<0.001. Error bars are SEM

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1000, HPA043867, Atlas Antibodies), SNX17 mouse mAb, VPS35L rabbit pAb (1:1000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1000, 11938-1-AP, Proteintech), GFP rabbit mAb (1:1000, Ab32146, Abcam), mouse/rabbit Integrin β1 goat pAb (1:1000, AF2405, R&D Systems) and GAPDH rabbit mAb (1:2000, 2118, Cell Signaling).

Techniques: Infection, Surface Biotinylation Assay, Knockdown, Western Blot, Control, shRNA, Two Tailed Test, Labeling, Immunostaining, Biomarker Discovery, Stable Transfection, Expressing, Transfection, Generated, Blocking Assay

Journal: bioRxiv

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity

doi: 10.1101/2023.02.20.529299

Figure Lengend Snippet: β1-integrin has roles in structural and functional plasticity during cLTP. (A) Diagram of experiment. DIV16-18 hippocampal neurons were treated with either β1-integrin blocking or isotype control antibodies for 30 min, followed by a 5 min cLTP stimulus. mEPSCs were recorded during the first 30 min after cLTP (cLTP<30) or from 30 to 90 min after cLTP (cLTP>30). (B) Examples of mEPSC recordings of neurons that were treated with isotype ctrl or β1-integrin blocking antibodies. Recordings were performed in the absence of cLTP (baseline), during the first 30 min after cLTP or from 30 to 90 min after cLTP. (C) Quantification of mEPSC amplitude. Isotype ctrl baseline: 12.240 ± 0.570, N=19 neurons; isotype ctrl cLTP<30min: 16.790 ± 1.106, N=9 neurons; isotype ctrl cLTP>30min: 16.900 ± 0.954, N=10 neurons; β1-integrin blocking baseline: 11.570 ± 0.373, N=9 neurons; β1-integrin blocking cLTP<30 min: 18.110 ± 1.247, N=6 neurons; β1-integrin blocking neurons cLTP>30 min: 13.170 ± 0.458, N=13 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****p<0.001. Error bars are SEM. (D) Quantification of mEPSC frequency. Isotype ctrl baseline: 1.191 ± 0.227, N=19 neurons; isotype ctrl cLTP<30min: 3.333 ± 0.800, N=9 neurons; isotype ctrl cLTP>30min: 3.110 ± 0.740, N=10 neurons; β1-integrin blocking baseline: 1.167 ± 0.296, N=9 neurons; β1-integrin blocking cLTP<30 min: 3.952 ± 1.214, N=6 neurons; β1-integrin blocking neurons cLTP>30 min: 1.096 ± 0.302, N=13 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, **p<0.01. Error bars are SEM. (E) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. Neurons were treated with ß1-integrin blocking or isotype control antibodies for 30 min, followed by a 5-min cLTP stimulus in the presence of antibodies where indicated. Neurons were further incubated in the presence of antibodies for 50 min before fixation. Scale bar, 5 µm. (F) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. Isotype ctrl: 0.626 ± 0.016, N=28 neurons; isotype ctrl with cLTP: 0.717 ± 0.018, N=25 neurons; β1-integrin blocking: 0.621 ± 0.015, N=31 neurons; β1-integrin blocking with cLTP: 0.628 ± 0.010, N=32 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***p<0.005. Error bars are SEM. (G)Quantification of dendritic spine density (spines/μm). Isotype ctrl: 1.044 ± 0.048, N=28 neurons; isotype ctrl with cLTP: 0.977 ± 0.050, N=25 neurons; β1-integrin blocking: 0.931 ± 0.054, N=31 neurons; β1-integrin blocking with cLTP: 0.931 ± 0.039, N=32 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1000, HPA043867, Atlas Antibodies), SNX17 mouse mAb, VPS35L rabbit pAb (1:1000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1000, 11938-1-AP, Proteintech), GFP rabbit mAb (1:1000, Ab32146, Abcam), mouse/rabbit Integrin β1 goat pAb (1:1000, AF2405, R&D Systems) and GAPDH rabbit mAb (1:2000, 2118, Cell Signaling).

Techniques: Functional Assay, Blocking Assay, Control, Transfection, Incubation

Journal: bioRxiv

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity

doi: 10.1101/2023.02.20.529299

Figure Lengend Snippet: Model of SNX17-mediated modulation of synaptic structure and function. The SNX17-Retriever pathway is required for dendritic spine maintenance and for the cLTP-dependent increase in dendritic spine size. Glycine-mediated cLTP (1) stimulates calcium entry through the NMDA receptor, which activates the CaMKII pathway (2). CaMKII activation is necessary and sufficient to promote the recruitment of SNX17 and the Retriever complex to dendritic spines (3), and activates the recycling of β1-integrin from endosomes to the plasma membrane (4). The surface levels of β1-integrin increase during cLTP and promote dendritic spine growth (5). Endosomal PI(3)P increases upon cLTP and may help with the recruitment of SNX17 to synapses. Created with BioRender.com.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1000, HPA043867, Atlas Antibodies), SNX17 mouse mAb, VPS35L rabbit pAb (1:1000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1000, 11938-1-AP, Proteintech), GFP rabbit mAb (1:1000, Ab32146, Abcam), mouse/rabbit Integrin β1 goat pAb (1:1000, AF2405, R&D Systems) and GAPDH rabbit mAb (1:2000, 2118, Cell Signaling).

Techniques: Activation Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Dual role of brain endothelial Gpr126 in blood-brain barrier development and ischemic stroke

doi: 10.1101/2022.09.09.507316

Figure Lengend Snippet: A. Representative immunoblotting for Gpr126 in cBECs isolated from WT pups at P18 transfected with nontargeting small-interfering RNA (siRNA; Ctrl [control]) or with siRNAs against Gpr126 (no. 13, no. 95, no. 97). Tubulin is shown as loading control. B, Gpr126/GAPDH ratio quantified by densitometry scanning and expressed as fold change, as shown in A. Each symbol represents a single experiment (n=12 WT mice for each independent experiment, as means ±SD). **, P ≤0.005; ****, P ≤0.00005 (Brown-Forsythe and Welch ANOVA, Dunnett’s T3 multiple comparison tests). C, Quantification of the relative gene expression of Gpr126 in cBECs isolated from WT pups at P18, as shown in A . Each symbol represents a single experiment (n=12 WT mice for each independent experiment, as means ±SD). *, P <0.05; Brown-Forsythe and Welch ANOVA, Dunnett’s T3 multiple comparison tests). D, Representative immunoblotting for total Creb and its phosphorylation (phospho-Creb s133) in cBECs isolated from P18 pups, transfected with nontargeting small-interfering RNA (siRNA; Ctrl [control]) or with siRNAs against Gpr126 (no. 97) and treated with Collagen IV (or PBS as vehicle) for 45’. Vinculin is shown as loading control. E, p-Creb s133/Creb ratio quantified by densitometry scanning and expressed as fold change, as shown in D . Each symbol represents a single experiment (n=12 WT mice for each independent experiment, as means ±SD). **, P <0.005; ***, P <0.0005, ****, P <0.00005 (Brown-Forsythe and Welch ANOVA, Dunnett’s T3 multiple comparison tests). F, Quantification of the relative gene expression of Gpr126 in cBECs isolated from pups at P18, as shown in D, except for the treatment with collagen IV for 24h. Each symbol represents a single experiment (n=12 WT mice for each independent experiment, as means ±SD). *, P <0.05; ns=not significant (Brown-Forsythe and Welch ANOVA, Dunnett’s T3 multiple comparison tests). G, Quantification of fold differences in Gpr126 an lrp1 expression in iBECs, following RT-qPCR analysis. (n=3, as means ±SD). ***, P <0.0005 (unpaired t-tests with Welch’s correction). H, Representative immunoblotting for α1-integrin, α3-integrin, β1-integrin, and myc in iBECs transfected with GFP and Gpr126-myc. The protein extracts (lysates) were immunoprecipitated with an anti-myc antibody [IPs (myc)] or IgG [IPs (IgG)] (only for Gpr126-myc cells), as control. Data are representative of 3 independent experiments.

Article Snippet: The antibodies used in this study were: anti-Gpr126 rabbit (1:500, ab75456; Abcam; WB; IF), anti-Gpr126 rabbit (1:10, Ab218046; Abcam; EM) anti-Gapdh mouse (1:1000, sc32233; Santa Cruz; WB), anti-podocalyxin goat (1:200, AF1556; R&D; IF), anti-tubulin mouse (1:2000, T9026; Sigma; WB), anti-phospho-Creb rabbit (1:1000, 9198; Cell Signaling; WB), anti-Creb rabbit (1:1000, 4820; Cell Signaling; WB), anti-myc mouse (1:1000, 2276; Cell Signaling; WB), anti-c-myc agarose affinity gel antibody rabbit (A7470; Sigma; IP), anti-Lrp1 mouse (1:1000; ab215997; Abcam; WB), anti-integrin β1 goat (1:200; AF2405; R&D; IP), anti-integrin β1 rabbit (1:1000; 4706; Cell Signaling; WB), rat anti-mouse β1-integrin (1:50, 553715; BD Biosciences, active β1, FACS); alexa Fluor 647 anti-mouse/rat β1-integrin (1:50, 102214; Biolegend, FACS); anti-integrin β1 (1:10, AF 2325; R&D Systems; EM), anti-integrin α1 goat (1:500; ab243032; Abcam; WB), anti-integrin α3 goat (1:3500; AF2787; R&D; WB), Horse anti-rabbit biotinylated IgG (1:400; VC-BA-1100-MM15; Vector Laboratories; IF), streptavidin Alexa Fluor 555 conjugate (1:400; S32355; Invitrogen; IF), anti-GFP chicken (1:1000; ab13970; Abcam; IF), anti-Pecam-1 goat (1:200; AF3628; R&D Systems; IF), and anti-isolectin-B4 (1:200; B-1205; Vector Laboratories; IF); anti-m/rCD31/PECAM-1 Alexa Fluor 488 Conjugated (1:50, FAB3628G; R&D Systems); Claudin-5 (1:200, Ab15106; Abcam; IHC) and Plvap (1:200, 550563 clone MECA-32; BD Biosciences, IHC); BUV805 Rat anti-Mouse CD3 molecular complex (Monoclonal; clone 17A2; BD Biosciences;1:200; Cat#741982, FACS).

Techniques: Western Blot, Isolation, Transfection, Small Interfering RNA, Expressing, Quantitative RT-PCR, Immunoprecipitation

Journal: bioRxiv

Article Title: Dual role of brain endothelial Gpr126 in blood-brain barrier development and ischemic stroke

doi: 10.1101/2022.09.09.507316

Figure Lengend Snippet: A, Representative immunoblotting for Lrp1, β1-integrin, and myc in immortalized brain endothelial cells (iBECs) transfected with Gpr126-myc. The protein extracts (lysates) were immunoprecipitated with an anti-myc antibody [IPs (myc)] or IgG [IPs (IgG)], as control. Data are representative of 3 independent experiments. B, Representative immunoblotting for β1-integrin and myc in iBECs transfected with Gpr126-myc. The protein extracts (lysates) were immunoprecipitated with an anti-β1-integrin antibody [IPs (β1-integrin)] or IgG as control. Data are representative of 3 independent experiments. C, EM of Gpr126 immunogold-labeled (10 nm gold, Gpr126 10 ) and β1-integrin (5 nm gold, β1-integrin 5 ) as representative cryo-sections of brain capillaries from WT mouse cortex at P18. PM, plasma membrane; E, endothelial cell; P, pericyte; LE, late endosome. Scale bars: 100 nm. D, E, Flow cytometric analysis of active and total β1-integrin-positive fBECs from WT and Gpr126 iECKO mice at P18. D , Active β1-integrin−/Pecam-1-positive cells ratio expressed as percentages. Each symbol represents a single experiment (n=3 WT and 3 Gpr126 iECKO mice for each independent experiment, as means ±SD). ***, P <0.0005 (unpaired t-tests with Welch’s correction). E, Total β1-integrin−/Pecam-1-positive cells ratio expressed as percentages. Each symbol represents a single experiment (n=3 WT and 3 Gpr126 iECKO mice for each independent experiment, as means ±SD). ns=not significant (unpaired t-tests with Welch’s correction). F, Schematic model for Gpr126 complex formation during angiogenesis. (a) Upon activation by collagen IV, Gpr126 binds to Lrp1 and α3β1-integrin (1). The complex goes through Lrp1-mediated endocytosis (2), which favours EC migration and angiogenesis. Then Lrp1 and Gpr126 are transported to the plasma membrane (3), through the recycling endosomes (4). (b) The absence of Gpr126 negatively affects BM deposition and expression of Lrp1. α3β1-integrin is not internalized, and hence it accumulates on the surface. These processes result in a reduction of migration and angiogenesis. BEC, brain endothelial cell; BM, basement membrane.

Article Snippet: The antibodies used in this study were: anti-Gpr126 rabbit (1:500, ab75456; Abcam; WB; IF), anti-Gpr126 rabbit (1:10, Ab218046; Abcam; EM) anti-Gapdh mouse (1:1000, sc32233; Santa Cruz; WB), anti-podocalyxin goat (1:200, AF1556; R&D; IF), anti-tubulin mouse (1:2000, T9026; Sigma; WB), anti-phospho-Creb rabbit (1:1000, 9198; Cell Signaling; WB), anti-Creb rabbit (1:1000, 4820; Cell Signaling; WB), anti-myc mouse (1:1000, 2276; Cell Signaling; WB), anti-c-myc agarose affinity gel antibody rabbit (A7470; Sigma; IP), anti-Lrp1 mouse (1:1000; ab215997; Abcam; WB), anti-integrin β1 goat (1:200; AF2405; R&D; IP), anti-integrin β1 rabbit (1:1000; 4706; Cell Signaling; WB), rat anti-mouse β1-integrin (1:50, 553715; BD Biosciences, active β1, FACS); alexa Fluor 647 anti-mouse/rat β1-integrin (1:50, 102214; Biolegend, FACS); anti-integrin β1 (1:10, AF 2325; R&D Systems; EM), anti-integrin α1 goat (1:500; ab243032; Abcam; WB), anti-integrin α3 goat (1:3500; AF2787; R&D; WB), Horse anti-rabbit biotinylated IgG (1:400; VC-BA-1100-MM15; Vector Laboratories; IF), streptavidin Alexa Fluor 555 conjugate (1:400; S32355; Invitrogen; IF), anti-GFP chicken (1:1000; ab13970; Abcam; IF), anti-Pecam-1 goat (1:200; AF3628; R&D Systems; IF), and anti-isolectin-B4 (1:200; B-1205; Vector Laboratories; IF); anti-m/rCD31/PECAM-1 Alexa Fluor 488 Conjugated (1:50, FAB3628G; R&D Systems); Claudin-5 (1:200, Ab15106; Abcam; IHC) and Plvap (1:200, 550563 clone MECA-32; BD Biosciences, IHC); BUV805 Rat anti-Mouse CD3 molecular complex (Monoclonal; clone 17A2; BD Biosciences;1:200; Cat#741982, FACS).

Techniques: Western Blot, Transfection, Immunoprecipitation, Labeling, Activation Assay, Migration, Expressing