lc3b  (Cell Signaling Technology Inc)

Journal: Pediatric Discovery

Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

doi: 10.1002/pdi3.70075

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

Techniques: Cell-Signaling

Journal: Pediatric Discovery

Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

doi: 10.1002/pdi3.70075

Figure Lengend Snippet: House dust mite (HDM) stimulation induces early growth response 1 (EGR1) expression and autophagy‐associated changes in airway epithelial cells. (A–C) EGR1 mRNA and protein levels in BEAS‐2B cells stimulated with HDM extract (30 μg/mL, 3 hours) or vehicle control, assessed by quantitative polymerase chain reaction (qPCR) and Western blot. (D–F) Western blot analysis of LC3B and p62 protein levels. The LC3B‐II/I ratio was quantified by densitometry. (G) Representative immunofluorescence images of LC3B puncta (green) in BEAS‐2B cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (H) IL‐33 release into culture supernatants measured by ELISA. (I) Airway resistance in response to increasing concentrations of methacholine in HDM‐challenged mice and controls. Data were analyzed using repeated‐measures ANOVA. (J) Representative hematoxylin and eosin‐stained lung tissue sections. Scale bar, 100 μm. (K–M) Immunofluorescence detection of EGR1 (red) and LC3B (green) in lung tissue sections. Nuclei were stained with DAPI (blue). Fluorescence intensity was quantified and normalized to the control. Data are presented as mean ± standard deviation. For in vitro experiments: (A, G) n = 3 independent experiments; (B–F) n = 4 independent experiments. For in vivo experiments: n = 4 mice per group. Statistical significance was determined by an unpaired Student's t ‐test unless otherwise indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Western Blot, Analysis, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Tissue, Fluorescence, Standard Deviation, In Vitro, In Vivo

Journal: Pediatric Discovery

Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

doi: 10.1002/pdi3.70075

Figure Lengend Snippet: Validation of early growth indicaton 1 ( EGR1 ) overexpression and knockdown efficiency, and the effects on autophagy‐related proteins and IL‐33 release in house dust mite (HDM)‐stimulated BEAS‐2B cells. (A–C) EGR1 mRNA and EGR1 protein levels in cells transfected with the EGR1 overexpression plasmid (oe‐EGR1) or empty vector, assessed by quantitative polymerase chain reaction (qPCR) and Western blot. (D–F) EGR1 mRNA and ERG1 protein levels in cells transfected with EGR1‐targeting siRNA (si‐EGR1) or scrambled control, assessed by qPCR and Western blot. (G–I) Western blot analysis of LC3B and p62 in oe‐EGR1‐transfected cells, followed by HDM stimulation (30 μg/mL, 3 h). The LC3B‐II/I ratio and p62 levels were quantified by densitometry. (J) IL‐33 release into culture supernatants under the same conditions, measured by ELISA. (K–M) Western blot analysis of LC3B and p62 in si‐EGR1‐transfected cells with HDM stimulation. (N) Corresponding IL‐33 release measured by ELISA. (O–P) Immunofluorescence detection of LC3B puncta (green) in oe‐EGR1 and si‐EGR1 cells stimulated with HDM. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are presented as mean ± standard deviation ( n = 3 independent experiments). Statistical significance for panels (A–P) was determined by one‐way ANOVA followed by appropriate post‐hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

Techniques: Biomarker Discovery, Over Expression, Knockdown, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, Analysis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Standard Deviation

Journal: Pediatric Discovery

Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

doi: 10.1002/pdi3.70075

Figure Lengend Snippet: PI3K‐III (Vps34) protein levels in response to house dust mite (HDM) and early growth response 1 (EGR1) manipulation, and the impact of 3‐methyladenine (3‐MA) on autophagy and IL‐33 release in EGR1‐overexpressing cells. (A–B) Western blot analysis and quantification of Vps34 protein levels in BEAS‐2B cells stimulated with HDM extract (30 μg/mL, 3 h) or vehicle control. (C–D) Western blot analysis of Vps34 protein levels in cells transfected with oe‐EGR1 or empty vector, with or without HDM stimulation. (E–F) Western blot analysis of Vps34 protein levels in cells transfected with si‐EGR1 or scrambled control, with or without HDM stimulation. (G–I) Western blot analysis of LC3B and p62 in oe‐EGR1‐transfected cells treated with or without Class III PI3K inhibitor 3‐methyladenine (3‐MA, 10 mmol/L, 6 h). (J) IL‐33 release in the same experimental setting, measured by ELISA. All data are presented as mean ± standard deviation ( n = 3 independent experiments). For panels (A–B), statistical significance was determined by an unpaired Student's t ‐test. For panels (C–J), one‐way ANOVA followed by appropriate post‐hoc tests was used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

Techniques: Western Blot, Analysis, Control, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Standard Deviation