anti elf2  (Proteintech)

Journal: bioRxiv

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1101/2025.02.27.640631

Figure Lengend Snippet: a, DNA sequence reveals the low levels of CpG sites in the promoter regions of mouse Tie2 and VE-cadherin (Cdh5). b , Volcano plot showing changes in methylation level of genes in Camkkβ +/+ and CamKKβ −/− mice. The ratio of CpG methylated sites in genes (+/– 2kb of the TSS) of Camkkβ +/+ /Camkkβ −/− mice were presented (fold changes vs . p-value). c , Quantified data showing hyper-methylation of Elf2 gene promoter in CamKKβ −/− mice. N = 3 samples per genotype; *p<0.05 (unpaired t test). d , DNA sequence analysis reveals the abundance of CpG islands and CpG sites in the promoter regions of human and mouse Elf2 genes. e , WT and Camkkβ −/− mice lung mRNA levels of Elf2 and Elf1 were determined by qRT-PCR. N = 3 mice per genotype; ns, not significant, ***p<0.001 (unpaired t test). f , DNA methyl transferase inhibition restores Elf2 expression in Camkkβ −/− mice. WT and Camkkβ −/− mice were injected with 5-AZA (1 mg/kg, i.p.) or vehicle for 5 days as described above in . Lungs were harvested and used for IB. N = 3 mice per group; representative blot is shown. g , Expression of WT-CaMKKβ in EC of Camkkβ −/− mice rescued Elf2 expression. Camkkβ −/− mice were injected with liposome-CaMKKβ plasmid (1 µg plasmid/g body weight) complexes containing either WT-CaMKKβ or the kinase defective CaMKKβ ( K193A -CaMKKβ) mutant. At 72 h after injection, lungs were harvested and used for IB analysis. N = 3 mice per group; representative blot is shown.

Article Snippet: Rabbit polyclonal antibody (pAb) against β-catenin (Cat # sc-7199), goat pAb against CaMKKβ (Cat # sc-9629), mouse mAb against Elf2/Nerf2 (Cat # sc-130632), mouse mAb against VE-cadherin/cdh5 (Cat # sc-9989), and goat pAb against vWF (Cat # sc-8068) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Sequencing, Methylation, Quantitative RT-PCR, Inhibition, Expressing, Injection, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1101/2025.02.27.640631

Figure Lengend Snippet: a , EC-specific disruption of Camkkβ in mice inhibits expression of Elf2, Tie2, and VE-cadherin. WT mice were injected with CRISPR plasmid encoding control-sgRNA (5′-GCGAGGTATTCGGCTCCGCG-3′) or m CaMKKβ-sgRNA (5′-AAACGTCGGATCAGCTTCTTTTTGC-3′). The liposomes and CRISPR plasmid mixture was injected i.v. into the mouse (1 µg plasmid/g body weight). Lungs harvested at 96 h after injection were used for IB analysis. Representative blots are shown (N = 4 mice/group). b-c , HLMVEC were transfected with 100 nM of either scrambled-siRNA (Sc-siRNA) or CaMKKβ-siRNA. At 72 h after transfection, cell lysates were used for IB analysis ( b ) or challenged with LPS (5 μg/ml) for the indicated times and then cells were stained with anti-VE-cadherin pAb and DAPI (scale bar = 20 μm) ( c ). b , Results show a representative blot. d-e , TLR4 signaling regulates endothelial expression of Tie2 and VE-cadherin through CaMKKβ-mediated Elf2 transcription. WT mice were challenged with LPS (10 mg/kg, i.p.) for different time intervals. After LPS challenge, lungs harvested were used for qRT-PCR ( d ) and IB analysis ( e ). e , results show a representative blot. d and e , Shown are mean values ± SEM (n =3 mice/time point). *p<0.05; **p<0.01; ****p<0.0001 (Two-way ANOVA).

Article Snippet: Rabbit polyclonal antibody (pAb) against β-catenin (Cat # sc-7199), goat pAb against CaMKKβ (Cat # sc-9629), mouse mAb against Elf2/Nerf2 (Cat # sc-130632), mouse mAb against VE-cadherin/cdh5 (Cat # sc-9989), and goat pAb against vWF (Cat # sc-8068) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Disruption, Expressing, Injection, CRISPR, Plasmid Preparation, Control, Liposomes, Transfection, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1101/2025.02.27.640631

Figure Lengend Snippet: a , HLMVEC incubated with serum free medium for 12 h were stained with anti-MeCP2 pAb and DAPI. b , HLMVEC incubated with serum free medium for 12 h and then incubated with and without CaMKKβ inhibitor STO-609 (1 μM) for 60 min. Thereafter cytosolic and nuclear fractions prepared were used for IB. c-d , HLMVEC pretreated with STO-609 (1 μM) or vehicle (DMSO) for 60 min were exposed to thrombin (25 nM) or LPS (5 μg/ml) for different time intervals and used for IB analysis to determine phosphorylation of MeCP2 at S421 and S80. Results shown are representative blots of 2 independent experiments. e , MeCP2 was associated with the Elf2 gene promoter under basal state in HLMVEC. HLMVEC grown to confluency were used for ChIP assay to determine MeCP2 interaction with the Elf2 promoter. Thrombin stimulation caused the dissociation of MeCP2 from the promoter regions of Elf2 gene. Results shown are mean values ± SEM of two experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (Two-way ANOVA). f-I , EC-restricted Mecp2 deletion ( Mecp2 1EC ) in mice augments expression of Elf2 and mitigated LPS-induced lung vascular leak. f , Lung endothelial cells (LEC) isolated from WT and Mecp2 1EC mice were used for IB analysis to determine the expression of MeCP2 and Elf2. Results showed the loss of MeCP2 and augmented expression of Elf2 in Mecp2 1EC mice LEC. g , Lungs harvested from WT and Mecp2 1EC mice were used for qRT-PCR to determine expression of mRNA for Elf2, VE-cadherin and Tie2. N = 3 mice/genotype; *p<0.05, ***p<0.001; ****p<0.0001 (unpaired t test). h, Lungs were used for IB to determine the expression of VE-cadherin and Tie2. i , WT and Mecp2 1EC mice were challenged with LPS (10 mg/kg; i.p.) and then used to assess in vivo lung vascular leak by measuring EBA uptake. N = 4 mice per genotype; **p<0.01; ****p< 0.0001 (Two-way ANOVA).

Article Snippet: Rabbit polyclonal antibody (pAb) against β-catenin (Cat # sc-7199), goat pAb against CaMKKβ (Cat # sc-9629), mouse mAb against Elf2/Nerf2 (Cat # sc-130632), mouse mAb against VE-cadherin/cdh5 (Cat # sc-9989), and goat pAb against vWF (Cat # sc-8068) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Staining, Expressing, Isolation, Quantitative RT-PCR, In Vivo

Journal: bioRxiv

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1101/2025.02.27.640631

Figure Lengend Snippet: a , Schematics showing Ets binding sites in the promoter regions of h Tie2 and h Cdh5. b-c , HLMVEC challenged with LPS (5 μg/ml) for different time intervals were used for ChIP assay to determine Elf2 binding to the promoter regions of Tie2 ( b ) and of VE-cadherin (Cdh5) ( c ). Results shown are mean values of three experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (One-way ANOVA). d , HLMVEC transfected with scrambled-siRNA (Sc-siRNA) or h Elf2-siRNA were used for IB analysis. e , HLMVEC transfected with Sc-siRNA or h Elf2-siRNA were stained with anti-VE-cadherin Ab and DAPI to assess endothelial AJ integrity (scale bar = 20 μm). f and g , HLMVEC transfected with Sc-siRNA or h Elf2-siRNA were used to measure PAR-1 activating peptide (25 μM)- or LPS (5 µg/ml)-induced changes in TER as an assessment of endothelial permeability. Arrow indicates time at which PAR-1 activating peptide (PAR-1 pep), LPS or media was added.

Article Snippet: Rabbit polyclonal antibody (pAb) against β-catenin (Cat # sc-7199), goat pAb against CaMKKβ (Cat # sc-9629), mouse mAb against Elf2/Nerf2 (Cat # sc-130632), mouse mAb against VE-cadherin/cdh5 (Cat # sc-9989), and goat pAb against vWF (Cat # sc-8068) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Transfection, Staining, Permeability

Journal: bioRxiv

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1101/2025.02.27.640631

Figure Lengend Snippet: EC-restricted deletion of Elf2 ( Elf2 ΔEC ) in adult mice reduces VE-cadherin and Tie2 expression and exacerbates sepsis-induced lung vascular injury. a, Depicts the protocol used to create EC-restricted Elf2 knockout ( Elf2 1EC ) mice. The mixture of liposome and plasmid expressing sgRNA to target mElf2 or scrambled sg-RNA (Sc-sgRNA) was injected i.v. into the CRISPR/Cas9-cdh5-Cre mice. Four days after injection, mice were used for experiments. b , LEC isolated from mice injected with Sc-sgRNA (WT) or sgRNA to target mElf2 (Elf2 1EC ) were used for IB analysis. c . IB analysis of lung tissue from WT and Elf2 1EC mice. d , lung sections from WT and Elf2 1EC mice were stained with antibodies specific to VE-cadherin, vWF (EC-marker), and DAPI. Right panels show the magnified images. e . H&E staining of lung sections. BR, bronchi; V, vessel. Green arrow heads showed increased perivascular space in Elf2 1EC mice indicating basal vascular leak. f , WT and Elf2 1EC mice were challenged with LPS (5 mg/kg, i.p.) and then used to assess in vivo lung vascular leak by measuring EBA uptake. Augmented lung vascular leak was observed in Elf2 1EC mice. N = 4 mice per genotype; ****p< 0.0001 (Two-way ANOVA). g , Survival following LPS (5 mg/kg, i.p.) in WT and Elf2 1EC mice. N = 6 in each group. **p< 0.01 (log-rank test). h , Model for epigenetic regulation of Elf2 gene expression in endothelial cells during repair of sepsis-induced lung vascular injury created in https://BioRender.com . In quiescent endothelial cells (EC), the CpG-rich promoter of the Elf2 gene is methylated by DNA methyltransferases (DNMTs). This methylation recruits the methyl-CpG binding protein MeCP2, which binds to the methylated CpG sites and represses Elf2 transcription. During endotoxemia, activation of TLR4 and/or PAR-1 increases intracellular Ca²⁺ levels in EC, leading to loss of endothelial barrier integrity through phosphorylation-driven internalization and ubiquitin-mediated degradation of VE-cadherin ( , ). The rise in intracellular Ca²⁺ also activates Ca²⁺/calmodulin-dependent kinase CaMKKβ, which translocates to the nucleus and phosphorylates MeCP2 at Ser 421 . This phosphorylation causes MeCP2 to dissociate from methylated CpG sites, triggering CpG demethylation and reactivation of Elf2 transcription. Once reactivated, Elf2 promotes its own expression and upregulates VE-cadherin and Tie2, facilitating the repair of the disrupted endothelial barrier. SAM, S-adenosyl-methionine; SAHC, S-adenosylhomocysteine; TLR4, toll-like receptor 4; PAR-1, protease-activated receptor 1; Thr, thrombin; orange star, methylated CpGs.

Article Snippet: Rabbit polyclonal antibody (pAb) against β-catenin (Cat # sc-7199), goat pAb against CaMKKβ (Cat # sc-9629), mouse mAb against Elf2/Nerf2 (Cat # sc-130632), mouse mAb against VE-cadherin/cdh5 (Cat # sc-9989), and goat pAb against vWF (Cat # sc-8068) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Knock-Out, Plasmid Preparation, Injection, CRISPR, Isolation, Staining, Marker, In Vivo, Gene Expression, Methylation, Binding Assay, Activation Assay

Journal: Advanced Science

Article Title: Cryoshocked Adipocytes Mediated Dual‐Modal Strategy Combining Photodynamic Therapy and Triptolide Palmitate for Pulmonary Metastatic Melanoma Treatment

doi: 10.1002/advs.202414307

Figure Lengend Snippet: Synergistic mechanism. a) Schematic illustration of the synergistic effect of Ce6‐pTP‐CsA in inducing apoptosis in A375 cells. b) Western blot analysis of CGRP78, PERK, P‐eIF2 α , ATF4, CHOP, and Cleaved‐Caspase 12 expression in A375‐M1 cells after treatment with PBS, pTP and Ce6‐pTP‐CsA (±hv) (n = 3). c) Western blot analysis of Bax, Bid, Cytochrome C, Bcl‐2, Cleaved‐Caspase 9, Cleaved‐Caspase 3, and Cleaved PARP expression in A375‐M1 cells after treatment with PBS, TP, pTP, Ce6‐CsA, pTP‐CsA, and Ce6‐pTP‐CsA (n = 3).

Article Snippet: The antibodies used for Western blotting, including FATP1, FABP4, CD36, CHOP, and ATF4, were obtained from Proteintech (IL, USA); P‐elF2 α and Bcl‐2 primary antibodies were from Abclonal (MA, USA); the PERK primary antibody was from Servicebio (Wuhan, China); and the Cleaved PARP, Cleaved‐Caspase 3, and Cleaved‐Caspase 9 primary antibodies were from CST (MA, USA).

Techniques: Western Blot, Expressing