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. 2015 Jun 8;10(6):e0129772. doi: 10.1371/journal.pone.0129772

Fig 2. Photolabeling and ATP binding characteristics of ABCB10 using 8-azido-ATP [γ] biotin.

Fig 2

A) SMPs (80 μg protein) from Control (-) or ABCB10-V5 (+) transfected cells were incubated with 0 or 50 μM of 8-azido-ATP [γ] biotin on ice for 5 min, treated with (+) or without (-) UV light for another 5 min and followed by IP with anti-V5 agarose affinity gel beads. After SDS-PAGE of the IP, membranes were blotted with ExtrAvidin-HRP (1:1000) or anti-ABCB10 antibody (1:2000). B) Representative Western blot shows that 8-azido-ATP [γ] biotin binding to ABCB10 was dose-dependent. Michaelis-Menten curve obtained from Western blot analyses is shown, with a Km value of 128.3 μM. Three independent experiments were conducted and data are shown as mean ± SEM.