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Alomone Labs asr 011
Asr 011, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/asr-011/pm40812162-64-26-27?v=Alomone+Labs
Average 93 stars, based on 5 article reviews

asr 011 - by Bioz Stars, 2026-07

93/100 stars

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Protocol of inhibiting DNMT3a using TF-3 (1 mg/kg , i.v ) in WT mice. After 1 h of TF-3 administration the mice received LPS (10 mg/kg, i.p ) and lungs were harvested after 8 h (n=5mice/group). DNMT activity (B) , mRNA expression of KLF2 (C) and lung edema (D) were determined (n=3). E, mRNA expression of indicated genes in control, FAK depleted, and FAK + DNMT3a-depleted EC. GAPDH was used as an internal control (n=3). F, Schematics showing generation of EC-Fak/Dnmt3a -/- (double knockout) mice. Offspring of Fak fl/fl and Dnmt3a fl/fl mice were bred with SCL-Cre ERT to generate Fak/Dnmt3a fl SCL CreERT mice. Tamoxifen (80 mg/kg, i.p) was injected into 4-week old Fak/Dnmt3a fl SCL- CreERT mice for five consecutive days followed by 7-day drug washout period. G, mRNA expression shows deletion of FAK and S1PR1 in lungs from double knockout versus control mice. Experiments were repeated three times independently. H, Lung edema was assessed by measuring lung weight-dry weight ratio in Fak fl/fl , EC-Fak -/- and EC-FAK-null mice receiving TF-3 (1 mg/kg, i.v). I, mRNA expression of indicated genes in lungs of Fak fl/fl , EC-Fak -/- and EC- Fak -/- /Dnmt3a -/- . GAPDH was used as an internal control (n=3). J-K, TEER assay in FAK- depleted EC without or with TF-3 (1 µM) treatment as described in J . Data in plots B, C, D, E, G, H, I and K show mean ± SEM. Statistical significance was assessed by One-way ANOVA followed by post hoc Tukey’s test for B, C, D, E, H, I and K, while unpaired t test was used for G . **** P < 0.0001 relative to siCtr (K), *** P < 0.001 relative to no treatment (C), Fak fl/fl (G, H), ** P < 0.01 to no treatment (B, D), siCtr (E) fl/fl (I), * P < 0.05 to siCtr (E), fl/fl (I). # P < 0.05 to LPS treated (B, D), ## P < 0.01 to no treatment (C) and ### P < 0.001 to EC-Fak -/- . Also see Supplementary Fig. 4.

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: Protocol of inhibiting DNMT3a using TF-3 (1 mg/kg , i.v ) in WT mice. After 1 h of TF-3 administration the mice received LPS (10 mg/kg, i.p ) and lungs were harvested after 8 h (n=5mice/group). DNMT activity (B) , mRNA expression of KLF2 (C) and lung edema (D) were determined (n=3). E, mRNA expression of indicated genes in control, FAK depleted, and FAK + DNMT3a-depleted EC. GAPDH was used as an internal control (n=3). F, Schematics showing generation of EC-Fak/Dnmt3a -/- (double knockout) mice. Offspring of Fak fl/fl and Dnmt3a fl/fl mice were bred with SCL-Cre ERT to generate Fak/Dnmt3a fl SCL CreERT mice. Tamoxifen (80 mg/kg, i.p) was injected into 4-week old Fak/Dnmt3a fl SCL- CreERT mice for five consecutive days followed by 7-day drug washout period. G, mRNA expression shows deletion of FAK and S1PR1 in lungs from double knockout versus control mice. Experiments were repeated three times independently. H, Lung edema was assessed by measuring lung weight-dry weight ratio in Fak fl/fl , EC-Fak -/- and EC-FAK-null mice receiving TF-3 (1 mg/kg, i.v). I, mRNA expression of indicated genes in lungs of Fak fl/fl , EC-Fak -/- and EC- Fak -/- /Dnmt3a -/- . GAPDH was used as an internal control (n=3). J-K, TEER assay in FAK- depleted EC without or with TF-3 (1 µM) treatment as described in J . Data in plots B, C, D, E, G, H, I and K show mean ± SEM. Statistical significance was assessed by One-way ANOVA followed by post hoc Tukey’s test for B, C, D, E, H, I and K, while unpaired t test was used for G . **** P < 0.0001 relative to siCtr (K), *** P < 0.001 relative to no treatment (C), Fak fl/fl (G, H), ** P < 0.01 to no treatment (B, D), siCtr (E) fl/fl (I), * P < 0.05 to siCtr (E), fl/fl (I). # P < 0.05 to LPS treated (B, D), ## P < 0.01 to no treatment (C) and ### P < 0.001 to EC-Fak -/- . Also see Supplementary Fig. 4.

Article Snippet: Anti-S1PR1 antibody (Cat #ASR-011) was acquired from Alomone Labs. Anti-Emerin (Cat #30853), anti-Src (Cat #2109), p-Src (Cat #12432), anti-Fak (Cat #3285T), anti-Lamin (Cat #86846S), anti- DNMT3a (Cat # 3598) antibodies from Cell Signaling Technology whereas, anti-actin (Cat #sc- 47778), anti-DNMT3b (Cat #sc-20704), anti-p-Tyr (PY99, Cat #sc-7020) and anti-p-Tyr (PY20, Cat # sc-508) were acquired from Santa Cruz Biotechnology.

Techniques: Activity Assay, Expressing, Control, Double Knockout, Injection

Volcano plot of differential genes from bulk RNA- seq analysis of EC sorted from Fak fl/fl and EC-Fak -/- mice lungs. S1PR1 mRNA (B) or protein (C) expression in FAK + versus FAK - EC. GAPDH was used an internal control for mRNA while actin was used as a loading control (n=3). D, Corresponding densitometry of the blots. E, S1P (1 mg/kg, i.v ) was administered to Fak fl/fl or EC-Fak -/- mice and after 30 min lung edema was assessed by measuring wet-to-dry weight ratio of the lungs (n=5 mice/group). F, A representative micrograph shows effect of S1P in re-assembling adherens junctions in control versus FAK-depleted EC. EC received 1 µM S1P for 15 min after which the cells were co-immunostained with anti-VE-cadherin and anti-FAK antibodies. Images were acquired using confocal microscope. Note, Control EC were plated at sub-confluent level to match gaps in FAK depleted EC before S1P stimulation (n=3). Scale bar 20 µm. G, Plot shows intercellular gap in control and FAK-depleted EC after with or without 1 µM S1P for 15 min. Gap area was assessed by ImageJ. The experiment was independently repeated three times. H, TEER in control and FAK-depleted EC at baseline and after addition of 1 µM S1P ( n = 8 wells/group). The assay was repeated three times independently. I-J, EC plated on TEER electrodes were processed as in H for 48 h. EC were then re-transfected with vector or GFP-tagged WT-S1PR1 cDNA and TEER was measured (I) and S1PR1 expression (J) was quantified after 72 h after addition of 1 µM S1P ( n = 8wells/group). The assay was repeated three times independently. Immunoblot of EC shows S1PR1 expression taking actin as a loading control (n=3). K, Edema measurement was performed in Fak fl/fl and EC-Fak -/- mice 48 h after delivery of liposomes containing VE-cadherin promoter driven GFP-tagged WT-S1PR1 cDNA as in 1K (n=6 mice/group). Data in plots B , D , E , G and K show mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’s test in E and K, while unpaired t-test was used in B, D and G. ** P < 0.01 relative to FAK fl/fl (B, D) and Fak fl/fl and EC-Fak -/- receiving empty vector (K) * P < 0.05 relative to siCtr (G). Also see Supplementary Fig. 5.

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: Volcano plot of differential genes from bulk RNA- seq analysis of EC sorted from Fak fl/fl and EC-Fak -/- mice lungs. S1PR1 mRNA (B) or protein (C) expression in FAK + versus FAK - EC. GAPDH was used an internal control for mRNA while actin was used as a loading control (n=3). D, Corresponding densitometry of the blots. E, S1P (1 mg/kg, i.v ) was administered to Fak fl/fl or EC-Fak -/- mice and after 30 min lung edema was assessed by measuring wet-to-dry weight ratio of the lungs (n=5 mice/group). F, A representative micrograph shows effect of S1P in re-assembling adherens junctions in control versus FAK-depleted EC. EC received 1 µM S1P for 15 min after which the cells were co-immunostained with anti-VE-cadherin and anti-FAK antibodies. Images were acquired using confocal microscope. Note, Control EC were plated at sub-confluent level to match gaps in FAK depleted EC before S1P stimulation (n=3). Scale bar 20 µm. G, Plot shows intercellular gap in control and FAK-depleted EC after with or without 1 µM S1P for 15 min. Gap area was assessed by ImageJ. The experiment was independently repeated three times. H, TEER in control and FAK-depleted EC at baseline and after addition of 1 µM S1P ( n = 8 wells/group). The assay was repeated three times independently. I-J, EC plated on TEER electrodes were processed as in H for 48 h. EC were then re-transfected with vector or GFP-tagged WT-S1PR1 cDNA and TEER was measured (I) and S1PR1 expression (J) was quantified after 72 h after addition of 1 µM S1P ( n = 8wells/group). The assay was repeated three times independently. Immunoblot of EC shows S1PR1 expression taking actin as a loading control (n=3). K, Edema measurement was performed in Fak fl/fl and EC-Fak -/- mice 48 h after delivery of liposomes containing VE-cadherin promoter driven GFP-tagged WT-S1PR1 cDNA as in 1K (n=6 mice/group). Data in plots B , D , E , G and K show mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’s test in E and K, while unpaired t-test was used in B, D and G. ** P < 0.01 relative to FAK fl/fl (B, D) and Fak fl/fl and EC-Fak -/- receiving empty vector (K) * P < 0.05 relative to siCtr (G). Also see Supplementary Fig. 5.

Techniques: RNA Sequencing Assay, Expressing, Control, Microscopy, Transfection, Plasmid Preparation, Western Blot, Liposomes

A, Schematic representation of S1PR1 promoter with three KLF2 binding sites (-341, -501, and -937 from TSS). B, EC were co-transfected with WT-S1PR1 luciferase promoter or mutated S1PR1 luciferase promoter (which lacks three KLF2 binding sites) along with KLF2 cDNA (n=3). Luciferase activity was determined after 24 h. C-D, EC were transfected with control shRNA or KLF2 shRNA. After 48 h, S1PR1 mRNA (C) or protein expression (D) was determined. GAPDH was used as a loading control for mRNA and actin was used as loading control for protein (n=3). E, Corresponding densitometry of the above blots. F, EC plated on TEER electrodes were transfected with control or KLF2 shRNA. After 48 h, cells were stimulated with S1P (1 µM) and TEER was measured as in 2G (n = 8wells/group). G, After 48 h of siFAK transfection, EC were co-transfected with vector or KLF2 cDNA. mRNA expression of indicated genes was assessed by qPCR. GAPDH was used an internal control (n=3). H-I, EC plated on TEER electrodes were transfected with control or FAK siRNA. After 48 h, cells were co-transfected with WT-KLF2 cDNA. After 72 h cells were stimulated with S1P, and TEER was assessed as in ( n = 8wells/group). J-K, S1PR1 mRNA expression in FAK-depleted EC after DNMT3a knockdown (J) or inhibition with 1 µM theaflavin-3,3’-digallate (TF-3) for 4 h. (K). L , S1PR1 mRNA in EC-Fak -/- mice after 24 h injection of AZA (0.5 mg/kg, i.v. ) or TF-3 (1mg/kg, i.v ). GAPDH was used an internal control (n=3). Data plotted in B, C, E, G, I, J, K and L are given as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’ test in B, G, I, J, K and L and unpaired t test was used in C and E. **** P < 0.0001 relative to siFAK+KLF2 (I), *** P < 0.001 to WT-S1PR1 (B), shCtr (C) Fak fl/fl (L), ** P < 0.01 relative to shCtr (E), siCtr (G, J) siCtr+TF-3 (K) and * P < 0.05 siCtr (G), siCtr+TF-3 (K). Also see Supplementary Fig. 5 .

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: A, Schematic representation of S1PR1 promoter with three KLF2 binding sites (-341, -501, and -937 from TSS). B, EC were co-transfected with WT-S1PR1 luciferase promoter or mutated S1PR1 luciferase promoter (which lacks three KLF2 binding sites) along with KLF2 cDNA (n=3). Luciferase activity was determined after 24 h. C-D, EC were transfected with control shRNA or KLF2 shRNA. After 48 h, S1PR1 mRNA (C) or protein expression (D) was determined. GAPDH was used as a loading control for mRNA and actin was used as loading control for protein (n=3). E, Corresponding densitometry of the above blots. F, EC plated on TEER electrodes were transfected with control or KLF2 shRNA. After 48 h, cells were stimulated with S1P (1 µM) and TEER was measured as in 2G (n = 8wells/group). G, After 48 h of siFAK transfection, EC were co-transfected with vector or KLF2 cDNA. mRNA expression of indicated genes was assessed by qPCR. GAPDH was used an internal control (n=3). H-I, EC plated on TEER electrodes were transfected with control or FAK siRNA. After 48 h, cells were co-transfected with WT-KLF2 cDNA. After 72 h cells were stimulated with S1P, and TEER was assessed as in ( n = 8wells/group). J-K, S1PR1 mRNA expression in FAK-depleted EC after DNMT3a knockdown (J) or inhibition with 1 µM theaflavin-3,3’-digallate (TF-3) for 4 h. (K). L , S1PR1 mRNA in EC-Fak -/- mice after 24 h injection of AZA (0.5 mg/kg, i.v. ) or TF-3 (1mg/kg, i.v ). GAPDH was used an internal control (n=3). Data plotted in B, C, E, G, I, J, K and L are given as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’ test in B, G, I, J, K and L and unpaired t test was used in C and E. **** P < 0.0001 relative to siFAK+KLF2 (I), *** P < 0.001 to WT-S1PR1 (B), shCtr (C) Fak fl/fl (L), ** P < 0.01 relative to shCtr (E), siCtr (G, J) siCtr+TF-3 (K) and * P < 0.05 siCtr (G), siCtr+TF-3 (K). Also see Supplementary Fig. 5 .

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Control, shRNA, Expressing, Plasmid Preparation, Knockdown, Inhibition, Injection

DNMT activity was measured in FAK depleted EC transduced with WT-emerin or phosphodefective emerin mutant after 24 h post-transfection as in (n=3). B, mRNA expression of KLF2 and S1PR1 in FAK depleted EC transducing WT-or phosphodefective-emerin. GAPDH was used as an internal control (n=3). C, Representative micrograph demonstrates intercellular gap as measured by immunostaining with anti-VE-Cadherin antibody in FAK-depleted EC after transduction with WT- or phosphodefective-emerin mutant. Scale bar 20 µm. D, Intercellular gap was measured as in 4F. E, EC plated on TEER electrodes were transfected with either Ctr or FAK siRNA and after 48h WT- or phosphodefective- emerin mutant was transduced. TEER was assessed after 72 h ( n = 8/group). The assay was repeated three times independently. Data plotted in A, B, D and E are given as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’s test. **** P < 0.0001 relative to siCtr (E), *** P < 0.001 siCtr (A, B), ** P < 0.01 siCtr (D), * P < 0.05 siCtr (D). F, Model of FAK suppression of nuclear mechanotransduction and synthesis of genes maintaining vascular barrier. We posit that loss of FAK increases RhoA activity which in turn increases intracellular mechanical tension by activating Rho kinase and myosin ATPase activity. Increased tension activates cSrc which phosphorylates emerin. Phosphorylated emerin activates DNMT3a, which in turn methylates the KLF2 promoter, blocking binding of its transcription factor, MEF2, and hence KLF2 synthesis. Suppression of KLF2 synthesis compromises S1PR1 transcription leading to conversion of restrictive EC to leaky EC which impairs vascular homeostasis.

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: DNMT activity was measured in FAK depleted EC transduced with WT-emerin or phosphodefective emerin mutant after 24 h post-transfection as in (n=3). B, mRNA expression of KLF2 and S1PR1 in FAK depleted EC transducing WT-or phosphodefective-emerin. GAPDH was used as an internal control (n=3). C, Representative micrograph demonstrates intercellular gap as measured by immunostaining with anti-VE-Cadherin antibody in FAK-depleted EC after transduction with WT- or phosphodefective-emerin mutant. Scale bar 20 µm. D, Intercellular gap was measured as in 4F. E, EC plated on TEER electrodes were transfected with either Ctr or FAK siRNA and after 48h WT- or phosphodefective- emerin mutant was transduced. TEER was assessed after 72 h ( n = 8/group). The assay was repeated three times independently. Data plotted in A, B, D and E are given as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’s test. **** P < 0.0001 relative to siCtr (E), *** P < 0.001 siCtr (A, B), ** P < 0.01 siCtr (D), * P < 0.05 siCtr (D). F, Model of FAK suppression of nuclear mechanotransduction and synthesis of genes maintaining vascular barrier. We posit that loss of FAK increases RhoA activity which in turn increases intracellular mechanical tension by activating Rho kinase and myosin ATPase activity. Increased tension activates cSrc which phosphorylates emerin. Phosphorylated emerin activates DNMT3a, which in turn methylates the KLF2 promoter, blocking binding of its transcription factor, MEF2, and hence KLF2 synthesis. Suppression of KLF2 synthesis compromises S1PR1 transcription leading to conversion of restrictive EC to leaky EC which impairs vascular homeostasis.

Techniques: Activity Assay, Transduction, Mutagenesis, Transfection, Expressing, Control, Immunostaining, Blocking Assay, Binding Assay

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