Research Paper Volume 9, Issue 4 pp 1248—1268

Ionizing radiation reduces ADAM10 expression in brain microvascular endothelial cells undergoing stress-induced senescence

Figure 4. Radiation modulates expression of the alpha-secretase, ADAM10. (A) Representative immunofluorescent images of ADAM10 expression 6 days after radiation (20 Gy) or sham treatment; ADAM10 (red), DAPI-stained nuclei (green); 100× magnification, n=2 independent experiments. Dotted lines in merged images indicate the boundary of several large senescent cells with reduced ADAM10 immunostaining. (B) Representative western blots of whole cell lysates (15 μg) probed for pro-ADAM10 and mature ADAM10. (C) Protein bands (ADAM10) were quantitated after normalization to GAPDH using Image J. Data represent mean ± SEM of 4 independent experiments. Student’s t-test **P<0.01. (DF) Representative images of fractionated extracts (10 μg each lane) from biotin-labelled cells before and after streptavidin enrichment: (D) Coomassie-stained SDS-PAGE gel; (E) streptavidin-HRP-probed membrane; (F) anti-ADAM10-probed membrane. IN, total cell extract prior to streptavidin enrichment; BB, biotin-bound fraction; OUT, non-biotinylated fraction eluted post-streptavidin binding; C, control; IR, irradiated.