rabbit polyclonal anti human hsp70 hspa1a  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation

doi: 10.1074/jbc.RA119.009145

Figure Lengend Snippet: Deletion of Hsp70.1/.3 attenuates airway inflammatory responses. A, Hsp70.1/.3+/+ (WT) and Hsp70.1/.3−/− (knockout) mice were sensitized and challenged with SEA or PBS at the indicated times to induce allergic airway inflammation. B, the gene expression of the heat shock proteins between the WT (filled circles and squares) and Hsp70.1/.3−/− mice (open circles and squares) in the lung tissues was measured by RT-PCR. The deletion of Hsp70 was confirmed in Hsp70.1/.3−/− mice (left), whereas the expression levels of Hsp40 (middle) and Hsp27 (right) were unaffected. C, 5-μm sections of paraffin-embedded lung tissues from the WT (top) and Hsp70.1/.3−/− (bottom) mice were stained with H&E. Saline- and SEA-treated lung sections are shown on the left and right, respectively; representative images are shown at 10× magnification. The airway inflammation in histological sections was quantified as peribronchial (D) and perivascular (E) inflammation scores. F, the lung sections were also stained with ABPAS for mucus hypersecretion. Representative images are shown as described in C, and images are shown at 20× magnification. G, mucus hypersecretion in ABPAS-stained sections was quantified with the mucus score. The quantitative data are shown as mean ± S.D. (error bars), n = 5–6 per group. Significant p values are presented as *, **, and **** for p < 0.05, p < 0.01, and p < 0.0001, respectively. Data in D and E are shown as pooled results from two repeated experiments with similar results (n = 10–15 mice/group); other figures are representative data of two repeated experiments. i.p., intraperitoneal; I.T., intratracheal.

Article Snippet: Paraffin-embedded sections (5 μm) were deparaffinized, extensively washed in PBS, blocked for 2 h with donkey serum, and incubated overnight at 4 °C with a rabbit anti-mouse Hsp70 polyclonal antibody (1:500 dilution; Thermo Fisher, Rockford, IL) in a humidified chamber.

Techniques: Knock-Out, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Saline

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation

doi: 10.1074/jbc.RA119.009145

Figure Lengend Snippet: Hsp70.1/.3−/− mice have decreased numbers of eosinophils and reduced levels of Th2 cytokines in the BAL fluid. BAL fluid was collected from WT (filled circles and squares) and Hsp70.1/.3−/− (open circles and squares) mice challenged with saline or SEA. A, the total BAL cell number was determined. B, the differential cell number in the BAL fluid was determined with Cytospin preparations. C–E, levels of Th2 cytokines IL-4 (C), IL-5 (D), and IL-13 (E) were measured by ELISA. The data are shown as the mean ± S.D. (error bars), n = 5–6 per group. Significant p values are shown as *, **, ***, and **** for p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Except for A and B shown as pooled data from two different experiments with similar results (n = 10–12 mice per group), data are shown as a representative of two different experiments with similar results (n = 5–6 mice/group).

Article Snippet: Paraffin-embedded sections (5 μm) were deparaffinized, extensively washed in PBS, blocked for 2 h with donkey serum, and incubated overnight at 4 °C with a rabbit anti-mouse Hsp70 polyclonal antibody (1:500 dilution; Thermo Fisher, Rockford, IL) in a humidified chamber.

Techniques: Saline, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation

doi: 10.1074/jbc.RA119.009145

Figure Lengend Snippet: Hsp70.1/.3 deficiency induces a decrease in gene expression of type 2 cytokines and a reduction of Th2 cytokine–producing T cells in the lungs. A–D, lung gene expression levels of the Th2 cytokines IL-4 (A), IL-5 (B), IL-13 (C), and IL-17 (D) were determined by quantitative RT-PCR. Immune cells were isolated from the lungs of WT and Hsp70.1/.3−/− mice and stimulated with phorbol 12-myristate 13-acetate and ionomycin for intracellular cytokine detection with FACS. The T cell populations were gated as CD3+ and CD4+ cells; the intracellular expression levels of IL-13 (E and F) and IL-17 (G and H) in CD4+ T cells were determined. The percentages (E and G) and MFI (F and H) of the CD4+ T cells expressing IL-13 or IL-17A are shown. The filled circles and squares represent the WT mice, and the open circles and squares represent the Hsp70.1/.3−/− mice. The data are shown as the mean ± S.D. (error bars), with n = 3–6 per group. One-way ANOVA was performed to compare differences between multiple groups. Significant p values are shown as ** and **** for p < 0.01 and p < 0.0001, respectively. Representative data of two different experiments are shown.

Article Snippet: Paraffin-embedded sections (5 μm) were deparaffinized, extensively washed in PBS, blocked for 2 h with donkey serum, and incubated overnight at 4 °C with a rabbit anti-mouse Hsp70 polyclonal antibody (1:500 dilution; Thermo Fisher, Rockford, IL) in a humidified chamber.

Techniques: Gene Expression, Quantitative RT-PCR, Isolation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation

doi: 10.1074/jbc.RA119.009145

Figure Lengend Snippet: Comparative analysis of up- or down-regulated genes in the lungs of WT and Hsp70.1/.3−/− mice during SEA-induced allergic airway inflammation. A, Venn diagram of the ratio of gene expression (SEA/saline) obtained in the lungs isolated from sensitized WT and Hsp70.1/.3−/− mice. Venn diagrams were obtained using raw data and filtering genes that were at least 1.5-fold up- or down-regulated when comparisons were made within each animal group and between the two following conditions: antigen challenge and saline challenge. B, the microarray data were analyzed by Ingenuity Pathway Analysis software, and the top three pathways of differential gene expression between the WT and Hsp70.1/.3−/− mice are shown. C–E, the heat maps present the expression change of genes involved in the agranulocyte adhesion and diapedesis (C), fibrosis (D), and granulocyte adhesion (E) pathways.

Article Snippet: Paraffin-embedded sections (5 μm) were deparaffinized, extensively washed in PBS, blocked for 2 h with donkey serum, and incubated overnight at 4 °C with a rabbit anti-mouse Hsp70 polyclonal antibody (1:500 dilution; Thermo Fisher, Rockford, IL) in a humidified chamber.

Techniques: Gene Expression, Saline, Isolation, Microarray, Software, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation

doi: 10.1074/jbc.RA119.009145

Figure Lengend Snippet: Hematopoietic BM-derived Hsp70 is responsible for airway inflammation in SEA-induced airway inflammation. A, immunohistochemistry staining of Hsp70 on paraffin-embedded lung sections from the WT mice challenged with SEA. The images are shown at low (10×) and high magnification (63×). The dashed box areas are shown at high magnification to highlight the infiltrating cells (arrows) during airway inflammation. Representative images are shown from five mice per group. The lung sections of chimeric mice were stained with H&E (B) and ABPAS (C), and the chimeric group details are shown above each image column. Representative images are shown at 10× and 20× magnification for H&E and ABPAS, respectively. The red arrows in C highlight the mucus staining in the bronchial epithelial cells. D, airway inflammation was quantified from the histological sections as the lung inflammation score (n = 4–6 mice/group). Data in D are shown as the mean +/− S.D. (error bars). One-way ANOVA was performed to compare differences between chimeric groups: *, p < 0.05. Representative data of two different chimeric assays with similar results are shown. KO, knockout.

Article Snippet: Paraffin-embedded sections (5 μm) were deparaffinized, extensively washed in PBS, blocked for 2 h with donkey serum, and incubated overnight at 4 °C with a rabbit anti-mouse Hsp70 polyclonal antibody (1:500 dilution; Thermo Fisher, Rockford, IL) in a humidified chamber.

Techniques: Derivative Assay, Immunohistochemistry, Staining, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 70 is a positive regulator of airway inflammation and goblet cell hyperplasia in a mouse model of allergic airway inflammation

doi: 10.1074/jbc.RA119.009145

Figure Lengend Snippet: Transfer of WT bone marrow to Hsp70.1/3-deficient mice restored airway inflammation and Th2 cytokine production. BALF was collected from chimeric mice challenged with SEA to induce allergic inflammation of the airways. The total numbers of BAL cells (A) and eosinophils (B) were counted. The Th2 cytokines IL-4 (C), IL-5 (D), and IL-13 (E) were quantified in BAL fluid using ELISA. Chimeric mouse details are shown on the bottom of each related figure. F, lung immune cells were prepared from the WT (filled squares) and Hsp70.1/.3−/− mice (open squares) sensitized and challenged with SEA and then stimulated with ConA or SEA or cultured with media alone. The cytokine levels of IL-13 were determined by ELISA. The data are shown as the mean ± S.D. (error bars). One-way ANOVA with multiple comparison test was used for the chimera assays in A–E, and an unpaired two-tailed Student's t test was used to compare IL-13 cytokine production between the WT and Hsp70.1/.3−/− mice in F. Significant p values are shown as *, **, and *** for p < 0.05, p < 0.01, and p < 0.001, respectively, with n = 4–6 per group. Pooled data from two different experiments are shown in A (n = 8–15); other figures are presented as representative data of two different experiments. KO, knockout.

Article Snippet: Paraffin-embedded sections (5 μm) were deparaffinized, extensively washed in PBS, blocked for 2 h with donkey serum, and incubated overnight at 4 °C with a rabbit anti-mouse Hsp70 polyclonal antibody (1:500 dilution; Thermo Fisher, Rockford, IL) in a humidified chamber.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Comparison, Two Tailed Test, Knock-Out