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Human anti Human DJ-1 antibody, clone 3055 used for the detection of oxidized DJ-1 by western blotting
Increased levels of DJ-1 and oxDJ-1 in RPE lysates and tissue from AMD donors. Lysates of human RPE isolated from non-AMD (A to C lanes 1 to 5) and AMD donors (A to C, lanes 6 to 10) were harvested and analyzed by immunoblot assay with DJ-1 antibody (A) and oxDJ-1 antibody (B). Each lane contained 20 µg of protein. Protein loads were confirmed in replicate blots probed with GAPDH (C). Immunoblots of lysates revealed that AMD RPE displayed significant increased immunoreactivity of both DJ-1 (A) and oxDJ-1 (B) when compared to non-AMD RPE lysates. Quantification of immunoblots demonstrated a 1.7 and 4 fold increase in the expression levels of DJ-1 and oxDJ-1, respectively (D). Blue columns = non-AMD; Red columns = AMD. Data is expressed as mean relative signal intensity ± SEM (n = 8). Asterisks denote statistical significance compared with non-AMD RPE (*p = p = 0.0098 for DJ-1 and **p = 0.0058 for oxDJ-1). Alternatively, cryosections of different non-AMD (E–H) and AMD (I to L) donors with geographic atrophy, and isolated Bruch’s membrane (BM) and choroid from two different AMD donors were (M to P) labeled with DJ-1 antibody. Negative control sections were reacted with DJ-1 antibody pre-absorbed with lysates of cells overexpressing DJ-1 and shoed no DJ-1 labeling (E, G, I, K, M, O). DJ-1 labeling was detected mostly in the RPE nuclei (arrowheads) but also in the cytoplasm of non-AMD donors (F and H, arrows). Significantly more DJ-1 was detected all over the cytoplasm of RPE cells from two different AMD donors (J and L, arrows), while DJ-1 was diffusely distributed in isolated BM and in drusen (E, G, asterisks). Scale bars (E to L) = 10 µm; (M to P) = 50 µm.
From: Shadrach KG, Rayborn ME, Hollyfield JG, Bonilha VL (2013) DJ-1-Dependent Regulation of Oxidative Stress in the Retinal Pigment Epithelium (RPE).
PLoS ONE 8(7): e67983.