Fig. 4.
Reversibility of pre-formed B2M-GAPDH cross-links. B2M (20 μM in 10 mM phosphate buffer, pH 7.4) was subjected to photo-oxidation for 60 min and then incubated with GAPDH (20 μM in 10 mM phosphate buffer, pH 7.4) for 1 h. Subsequently the cross-linked samples were subjected to SDS-PAGE under non-reducing (panel A and B, lanes 1–3) or reducing condition (panel A and B, lanes 5–7), or incubated with DTT (2 mM in 10 mM phosphate buffer, pH 7.4; panels D and E, lanes 6–8) or TCEP (2 mM in 10 mM phosphate buffer, pH 7.4; panels D and E, lanes 9–11) for a further 1 h before analysis by immunoblotting. Panels A and D: detection with the anti-B2M antibody. Panels B and E: as panels A and D, respectively, but with detection using an anti-GAPDH antibody. Panel C: Optical density ratio (ODlane 7/ODlane 3) of cross-linked bands (~48k Da) from immunoblotting data in panels A and B. Panel F: Optical density ratio (ODlane 8 or lane 11/ODlane 5) of cross-linked bands (~48k Da) from immunoblotting data in panels D and E. Each blot is representative of one of three, obtained from three independent experiments. Quantitative data are presented as mean ± SD from three independent experiments. Statistical differences were examined using a Student's t-test and are indicated as follows: *P < 0.05 for lane 7 vs. lane 3 in panels A and B. #P < 0.05 vs lane 5 in panels D, E.