DBP carbonylation impedes DBP-mediated inhibition of 25(OH)D3-induced CD38 expression. (A) Western blot of DBP and albumin in serial dilutions of plasma and central lymph. (B) Western blot of non-oxidized (lanes 1 and 2) and oxidized (lanes 3 and 4) DBP that subsequently had been derivatized with 2,4-dinitrophenyl hydrazine to detect carbonyl groups (lanes 2 and 4) or left untreated (lanes 1 and 3). The membranes were first analysed with anti-DNP antibodies to detect carbonylated DBP. Subsequently, the membranes were stripped and re-blotted with anti-DBP antibodies to detect total DBP. (C) Relative CD38 cell surface expression on T cells activated in the presence of 25(OH)D3 (100 nM) and the indicated concentrations of DBP or carbonylated DBP (CarboDBP). Mean values ± SEM from 3 independent experiments are plotted. (D) Representative Western blot analysis of carbonylated DBP and total DBP (loading control) in human serum. Precipitated DBP was either derivatized with 2,4-dinitrophenyl hydrazine to detect carbonyl groups (lanes 1 and 2) or left untreated (lanes 3 and 4). Lanes 1 and 3 present precipitated DBP diluted 1:4, and lanes 2 and 4 present precipitated DBP diluted 1:8.