primary antibody against apn  (R&D Systems)

Journal: Aging and Disease

Article Title: Adiponectin mRNA Conjugated with Lipid Nanoparticles Specifically Targets the Pathogenesis of Type 2 Diabetes

doi: 10.14336/AD.2024.0162

Figure Lengend Snippet: APN in situ expression . ( A ) APN gene expression in different tissues. Skeletal muscles; Liver; Kidney; White fats; Brown fats. APN gene expression was calculated based on the Law of Fold-change, which is 2 -ΔΔCT . Fold-change values less than one indicate downregulation, whereas values greater than one indicate upregulation. In vivo , a total of five biological samples (n = 5) were collected. Of each sample, three replicates were performed technically., ** P < 0.01 vs. PBS control group and ## P <0.01 vs. Empty-LNP group. ( B ) Immunohistochemical staining for adiponectin in T2D skeletal muscles, liver, and kidney mice in the studied groups. Negative controls were performed by blocking PBS without APN-Ab. Treated groups were performed by blocking with APN-Ab (200X). Labeling index was used to determine APN-Ab percentile expression (the ratio of positively stained cells/total cells × 100). Blinded staining intensity scores were assigned to each specimen based on arbitrary values of 1, 2, or 3 (reflecting weak, intermediate, and bright staining). A total of five biological samples (n = 5) were collected; three sections were taken from each sample. Scale bar= 50 μm.

Article Snippet: Protein Tech supplied the adiponectin primary antibody (1:400; #21613-1-AP, Rosemont, IL, USA).

Techniques: In Situ, Expressing, Gene Expression, Muscles, In Vivo, Control, Immunohistochemical staining, Staining, Blocking Assay, Labeling

Journal: Aging and Disease

Article Title: Adiponectin mRNA Conjugated with Lipid Nanoparticles Specifically Targets the Pathogenesis of Type 2 Diabetes

doi: 10.14336/AD.2024.0162

Figure Lengend Snippet: Conclusion based on our hypothesis . The alpha and beta cells of the islet of Langerhans work together to control and regulate glucose uptake, gluconeogenesis, and lipolysis in normal conditions. There are several abnormal features associated with type 2 diabetes, including increased adiposity, inflammation, insulin resistance, and decreased adipose tissue function. Thus, adiponectin (APN) production is reduced. As a result of the inhibition of adiponectin receptors (AdipoR1/AdipoR2) and subsequent association with adapter proteins, glucose uptake is inhibited by APPL1-Rab5 or APPL1-AMP-AMPK-mediated translocation of glucose transporter 4 (GLUT4), resulting in an activation of insulin resistance signaling . Insulin resistance is commonly characterized by decreased GLUT4-dependent glucose absorption in skeletal muscles and adipose tissue . A temporal increase in intracellular diacylglycerol (DAG) mass is associated with glucose-induced insulin resistance . Prolonged elevation of intracellular DAG activates protein kinase C (PKC) isoforms, leading to insulin resistance, intracellular lipid accumulation, and impaired signal transmission [ , ]. Glucose transport is downregulated due to increased PKC-mediated serine phosphorylation of the insulin receptor (IR) and insulin receptor substrate 1 (IRS-1) [ , ]. Controlling hyperglycemia can reverse the decline in diacylglycerol kinase delta (DGKd) protein and DGK kinase activity . Moreover, insulin resistance and diabetic nephropathy (DN) in T2D are associated with epidermal growth factor receptor (EGFR) activation. This activation increases immune cell infiltration and oxidative stress in the kidney and adipose mass while simultaneously reducing pancreatic insulin synthesis and adipocyte adiponectin production . Inhibition of EGFR decreases the expression of proinflammatory cytokines (iNOS, TNF-α, INF-γ, IL-6) . As a result of the administration of APN-mRNA-LNP, we have found that all previous targets have been successfully corrected. The result was improvements in several aspects of diabetes pathogenesis, including glucose uptake, insulin resistance, inflammation, and diabetic complications.

Article Snippet: Protein Tech supplied the adiponectin primary antibody (1:400; #21613-1-AP, Rosemont, IL, USA).

Techniques: Control, Inhibition, Translocation Assay, Activation Assay, Muscles, Transmission Assay, Phospho-proteomics, Activity Assay, Expressing

Journal: Diabetes

Article Title: Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes.

doi: 10.2337/db23-0455

Figure Lengend Snippet: Figure 1—Human GEnCs express AdipoRs and respond to adiponectin in vitro. A) Representative Western blots of AdipoR1 and AdipoR2 demonstrating kidney lysates with adipocytes as a positive control and b-actin as a loading control. Aii) Densitometry of four Western blots showing AdipoR1 and AdipoR2 protein level normalized to b-actin. B) qPCR analysis graph representing the relative mRNA expression of AdipoR1 and AdipoR2 across kidney tissue, glomerular cells, and adipocytes (n = 4 repeats each in triplicate). Data are plotted as the mean 2-(DCT) of each triplicate with mean (normalised to housekeeping gene). Ci) Representative Western blot demonstrating gAd (at 2.5 mg/mL) or vehicle effects at 30 min in CiGEnCs on AMPK, ACC, p-38, and Akt phosphorylation (p-) and total (T-) proteins (duplicates are shown). Cii) Densitometry confirmed the phosphorylation of AMPK, ACC, p-38, and Akt in response to gAd at 30 min compared with controls. Densitom- etry was performed on four representative blots from three independent repeats showing levels of phosphorylated protein of interest normal- ized to b-actin loading control. Bars/dots represent means ± SEM, one-way ANOVA *P < 0.05, **P < 0.01, post hoc analysis (Bonferroni). Ctrl, control; Gloms, glomeruli.

Article Snippet: An individual Table 2—Primary and secondary antibodies Antibody type Name Source Molecular weight (kDa) Dilution Validation Supplier; catalog no. Research resource identifier Primary p-AMPKa (Thr172) Rabbit 62 1:1,000 CST CST; 2535 AB_331250 Primary p-ACC (Ser79) Rabbit 280 1:1,000 CST CST; 11818 AB_2687505 Primary p-Akt (Ser473) (D9E) Rabbit 62 1:1,000 CST CST; 4060 AB_2315049 Primary p-P38MAPK Rabbit 43 1:1,000 CST CST; 4511 AB_2315049 Primary AdipoR1 (EPR6626) Rabbit 44 1:1,000 Human kidney lysate Abcam; antibody 126611 AB_11129655 Primary AdipoR2 Rabbit 43 1:1,000 Human kidney lysate ThermoFisher; 36-3100 AB_2849888 Primary b-actin Mouse 42 1:1,000 NA Sigma; A5441 AB_476744 Primary MMP2 60 1:1,000 Human kidney lysate Proteintech; 10373-2-AP AB_2919317 Secondary Goat anti-mouse Goat 1:500 Life Technologies UK; A11001 AB_2534069 Secondary Goat anti-rabbit Goat 1:500 Life Technologies UK; A11008 AB_143165 D ow nloaded from http://diabetesjournals.org/diabetes/article-pdf/doi/10.2337/db23-0455/751945/db230455.pdf by Viet N am Institution user on 11 April 2024 glomerulus was trapped on a custom-made petri dish and the perfusate was switched from 30 mg/mL labeled Alexa Fluor 488–BSA to 30 mg/mL unlabeled BSA.

Techniques: In Vitro, Western Blot, Positive Control, Control, Expressing, Phospho-proteomics

Journal: Diabetes

Article Title: Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes.

doi: 10.2337/db23-0455

Figure Lengend Snippet: Figure 2—AdipoR1 is important for adiponectin-mediated signaling in GEnCs. AdipoR1 knockdown was carried out using shRNA. A) Re- duced AdipoR1 mRNA expression (gene of interest/housekeeping gene; 2-(DCT) was confirmed by three separate shRNA constructs in GEnCs (n = 4). One-way ANOVA; *P < 0.05, **P < 0.01. B) AdipoR2 mRNA expression (gene of interest/housekeeping gene; 2-(DCT)) was not significantly affected. C) Representative Western blot demonstrating the knockdown extent of AdipoR1 in shRNA v726. D) Densitome- try confirmed the knockdown of AdipoR1 protein expression in CiGEnCs., Data are normalized to the b-actin loading control. Scattered dots represent means ± SEM, n = 3; unpaired t test; *P < 0.05. E) Representative Western blot demonstrating the effects of gAd (2.5 mg/mL) on scrambled controls, and AdiporR1 knockdown (R1 KD) on AMPK and ACC phosphorylation in CiGEnCs. F and G) Densitometry showing the extent of phosphorylation of AMPK and ACC in the three cell clones after 30 min of gAd stimulation. Data are normalized to the b-actin loading control. Dots represent means ± SEM, n = 4. One-way ANOVA; **P < 0.01, ***P < 0.001, ****P < 0.0001 by post hoc analysis (Bonferroni).

Article Snippet: An individual Table 2—Primary and secondary antibodies Antibody type Name Source Molecular weight (kDa) Dilution Validation Supplier; catalog no. Research resource identifier Primary p-AMPKa (Thr172) Rabbit 62 1:1,000 CST CST; 2535 AB_331250 Primary p-ACC (Ser79) Rabbit 280 1:1,000 CST CST; 11818 AB_2687505 Primary p-Akt (Ser473) (D9E) Rabbit 62 1:1,000 CST CST; 4060 AB_2315049 Primary p-P38MAPK Rabbit 43 1:1,000 CST CST; 4511 AB_2315049 Primary AdipoR1 (EPR6626) Rabbit 44 1:1,000 Human kidney lysate Abcam; antibody 126611 AB_11129655 Primary AdipoR2 Rabbit 43 1:1,000 Human kidney lysate ThermoFisher; 36-3100 AB_2849888 Primary b-actin Mouse 42 1:1,000 NA Sigma; A5441 AB_476744 Primary MMP2 60 1:1,000 Human kidney lysate Proteintech; 10373-2-AP AB_2919317 Secondary Goat anti-mouse Goat 1:500 Life Technologies UK; A11001 AB_2534069 Secondary Goat anti-rabbit Goat 1:500 Life Technologies UK; A11008 AB_143165 D ow nloaded from http://diabetesjournals.org/diabetes/article-pdf/doi/10.2337/db23-0455/751945/db230455.pdf by Viet N am Institution user on 11 April 2024 glomerulus was trapped on a custom-made petri dish and the perfusate was switched from 30 mg/mL labeled Alexa Fluor 488–BSA to 30 mg/mL unlabeled BSA.

Techniques: Knockdown, shRNA, Expressing, Construct, Western Blot, Control, Phospho-proteomics, Clone Assay

Journal: Journal of Inherited Metabolic Disease

Article Title: Skin inflammation and impaired adipogenesis in a mouse model of acid ceramidase deficiency

doi: 10.1002/jimd.12552

Figure Lengend Snippet: Impaired differentiation of Asah1 P361R/P361R preadipocytes accompanied by activation of inflammatory pathways. Impaired adipogenesis of adipose‐derived stem/stromal cell (ASC) from Asah1 P361R/P361R mice visualized by reduced Oil Red O and adiponectin in mature Asah1 P361R/P361R ASC‐derived adipocytes (ASC‐A) in comparison to wild‐type (WT). Asah1 P361R/P361R derived ASC‐A showed decreased Oil Red O and CatD staining (A). Asah1 P361R/P361R derived fibroblasts appeared to have similar Oil Red O and CatD staining and normal differentiation (B). Representative immunoblots of total and phosphorylated inflammatory proteins signal transducer and activator of transcription 3 (STAT3), NF‐κB, and JAK in ASC and ASC‐A homogenates from WT and Asah1 P361R/P361R mice (C) and their quantification relative to WT mice (D). Monocyte chemotactic protein‐1 (MCP‐1) and IL‐6 released in ASC and ASC‐A cell culture media collected during adipogenesis assessed by ELISA (E; n = 3 mice per group). Scale bars (A,B) = 100 μm

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies CatD (cn: ab75852, Abcam) and mouse Adiponectin (cn: BS8216211, MyBioSource, San Diego, California) diluted in blocking buffer.

Techniques: Activation Assay, Derivative Assay, Comparison, Staining, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay