Identification of hNopp140 as a Binding Partner for Doxorubicin with a Phage Display Cloning Method  Youngnam Jin, Jaehoon Yu, Yeon Gyu Yu  Chemistry & Biology  Volume 9, Issue 2, Pages 157-162 (February 2002) DOI: 10.1016/S1074-5521(02)00096-0

Figure 1 Structure of Doxorubicin and Biotinylated Doxorubicin The amine group of doxorubicin was coupled with NHS-biotin to produce biotinylated doxorubicin. Chemistry & Biology 2002 9, 157-162DOI: (10.1016/S1074-5521(02)00096-0)

Figure 2 Analysis of Phages Eluted after Each Round of Biopanning (A) The phage titer was determined for each washing and eluted solution at each round. (B) The insert size of the phage DNA from the whole eluted phage sample at each round was analyzed by polymerase chain reaction (PCR) with primers flanking the insertion sites. The PCR products from the first-, second-, third-, and fourth-round elutions were loaded in lanes 1, 2, 3, and 4, respectively, along with marker DNA (lane M). Chemistry & Biology 2002 9, 157-162DOI: (10.1016/S1074-5521(02)00096-0)

Figure 3 Phosphorylation of the Purified Trx-hNopp140C (A) The native form of Trx-hNopp140C (lane 1), along with the dephosphorylated (lane 2) and phosphorylated (lane 3) forms, was analyzed by 12% SDS-gel electrophoresis. (B) When 32P-d-ATP was used in the phosphorylation reaction, Trx-hNopp140C was labeled with 32P. (C) Dephosphorylated (lane 1), native (lane 2), and phosphorylated forms (lane 3) of Trx-hNopp140C were analyzed by 8% Urea-PAGE. The marked band in lane 2 of panel (A) and in lane 1 of panel (C) was calf intestine phosphatase (dephosphorylating enzyme). Chemistry & Biology 2002 9, 157-162DOI: (10.1016/S1074-5521(02)00096-0)

Figure 4 Phosphorylation-Dependent Aggregation of Trx-hNopp140C The formation of insoluble aggregates of Trx-hNopp140C was examined after incubation with 25 mM each of NaF and MgCl2 at 4°C for 1 hr. Native (lane 1 and 2) or phosphorylated Trx-hNopp140C (lanes 3 and 4) was incubated in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of NaF and MgCl2, and the soluble fraction was analyzed by 12% SDS-PAGE. Chemistry & Biology 2002 9, 157-162DOI: (10.1016/S1074-5521(02)00096-0)

Figure 5 Fluorescence Spectra of Doxorubicin and Trx-P140C/Doxorubicin Complex The fluorescence spectrum of 0.7 μM doxorubicin alone (bold line) in 10 mM Mops buffer containing 150 mM NaCl and 0.5 mM EDTA (pH 7.5) was compared with the spectrum of doxorubicin in the presence of 10 μM native (thin line) or phosphorylated forms of Trx-hNopp140C (dotted line). The excitation wave-length was 490 nm, and the emission spectrum was obtained between 530 and 600 nm. Chemistry & Biology 2002 9, 157-162DOI: (10.1016/S1074-5521(02)00096-0)

Figure 6 The Binding Sensorgram of the C-Terminal Region of hNopp140 on Doxorubicin Sensorgrams were obtained for 62.5 nM (thin line), 1 μM (dotted line), and 2 μM (bold line) of Trx-hNopp140C, 4 μM of phosphorylated Trx-hNopp140C (broken line), and 100 nM of DNA (bold broken line) against doxorubicin immobilized on the sensor chip. Chemistry & Biology 2002 9, 157-162DOI: (10.1016/S1074-5521(02)00096-0)