anti rage blocking antibody  (R&D Systems)

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly

doi: 10.1016/j.matbio.2016.07.003

Figure Lengend Snippet: Mesangial cells were grown for 2 days on tissue culture plastic + AGE-BSA or BSA (20 µg/mL) as in Figure 5 legend. 10 µg/mL rat plasma FN and 15 µg/ml RAGE function-blocking antibody were added for the final 24 hours. (A) Phase and fluorescence images were captured as described in Figure 5 legend. Average FN fluorescence intensity was decreased 1.16-fold to 0.86 ± range (n = 2) in the presence of RAGE function-blocking antibody with AGE-BSA compared to AGE-BSA treatment alone. Scale bar is 200 µm. (B) The DOC-insoluble fraction was separated by SDS-PAGE and immunoblotted with IC3 anti-rat FN monoclonal. Relative densitometry values are the mean of two experiments ± range normalized to GAPDH from the DOC soluble fraction. Blot is representative of two independent experiments.

Article Snippet: 15 μg/ml RAGE function blocking antibody (AF1179, R&D Systems, Minneapolis, MN) or lysyl oxidase (LOX) antibody (ab31238, Abcam, Cambridge, MA) was added for final 24 hours of 48 hour time-course where indicated.

Techniques: Clinical Proteomics, Blocking Assay, Fluorescence, SDS Page

Journal: PloS one

Article Title: HMGB1 accelerates alveolar epithelial repair via an IL-1β- and αvβ6 integrin-dependent activation of TGF-β1.

doi: 10.1371/journal.pone.0063907

Figure Lengend Snippet: Figure 1. Endogenous HMGB1 released by damaged primary rat ATII cells increases alveolar epithelial wound closure. (A) HMGB1 was elevated in cell supernatant from rat ATII monolayers that underwent scratch wounds (MS Cell Sup) compared to cell supernatant from rat ATII monolayers that did not undergo scratch wounds (condition media). (B) MS Cell Sup increases the rate of wound closure of primary rat ATII cell monolayers compared to cell supernatant from rat ATII monolayers that did not undergo scratch wounds. HMGB1 was depleted from MS Cell Sup by immunoprecipitation using 30 mg/ml of HMGB1 specific Ab (MS Cell Sup IP w/HMGB1 Ab). Controls were MS Cell Sup immunoprecipitated with a control IgG (MS Cell Sup IP w/Cont Ab). (C) HMGB1 is secreted by primary rat ATII cell monolayers after scratch wounds. Multiple scratches (MS) were performed on primary rat ATII cell monolayers. Fresh cell media were added for 6 hours to the monolayers after extensive washes. Cell supernatants were then centrifuged to remove dead cells and cell debris, then analyzed by western blot (40 ml loaded per lanes from a 1 ml MS Cell Sup sample). (D) MS Cell Sup increases the rate of wound closure of a primary rat ATII cell monolayers via RAGE- and TLR4-dependent pathways, but not via a CXCR4-dependent mechanism. MS Cell Sup, and either 30 mg/ml of blocking RAGE or TLR4 antibodies or their isotype control IgG, or 1 mM of AMD3100, a CXCR4 inhibitor, were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *p,0.05 from monolayers exposed to control cell media; **p,0.05 from monolayers exposed to MS Cell Sup. For western blot experiments, one representative experiment is shown, three additional experiments gave comparable results; *p,0.05 from monolayers exposed to condition media. doi:10.1371/journal.pone.0063907.g001

Article Snippet: Rat RAGE blocking antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Control, Western Blot, Blocking Assay