Sepharose 4b

Thiacremonone was conjugated with cyanogen bromide (CNBr)-activated Sepharose 4B (Sigma-Aldrich, St. Louis, MO). Briefly, thiacremonone (1 mg) was dissolved in 1 ml of coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl, pH 6.0). The CNBr-activated Sepharose 4B was swelled and washed in 1 mM HCl through a sintered glass filter, then washed with the coupling buffer. CNBr-activated Sepharose 4B beads were added to the thiacremonone-containing coupling buffer and incubated at 4°C for 24 hr. The thiacremonone-conjugated Sepharose 4B was washed with three cycles of alternating pH wash buffers (buffer 1, 0.1 M acetate and 0.5 M NaCl, pH 4.0; buffer 2, 0.1 M Tris-HCl and 0.5 M NaCl, pH 8.0). Thiacremonone-conjugated beads were then equilibrated with a binding buffer (0.05 M Tris-HCl and 0.15 M NaCl, pH 7.5). The control unconjugated CNBr-activated Sepharose 4B beads were prepared as described above in the absence of thiacremonone. The cell lysate or PRDX6 recombinant protein (Abnova, Taipei, Taiwan) were mixed with thiacremonone-conjugated Sepharose 4B or Sepharose 4B at 4°C for 24 hr. The beads were then washed three times with TBST. The bound proteins were eluted with SDS loading buffer. The proteins were then resolved by SDS-PAGE followed by immunoblotting with antibodies against PRDX6 (1∶1,000 dilution, Abcam, plc. Cambridge, UK).

KRICT-9 was combined with cyanogen bromide (CNBr)-activated Sepharose 4B (Sigma). Coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl, pH 6.0) was used to dissolve KRICT-9 (1 mg). CNBr-activated Sepharose 4B was washed in 1 mM HCl with coupling buffer on a sintered glass filter. Then it was added to KRICT-9 with same buffer at 4°C for 24 h. Conjugated KRICT-9 with CNBr-activated Sepharose 4B was washed with three cycles of alternating pH wash buffers (buffer 1, 0.1 M acetate and 0.5 M NaCl, pH 4.0; buffer 2, 0.1 M Tris HCl and 0.5 M NaCl, pH 8.0). Conjugated KRICT-9 was then equilibrated with binding buffer (0.05 M Tris HCl and 0.15 M NaCl, pH 7.5). The control group, which was not combined with CNBr-activated Sepharose 4B beads, was prepared in the same way. KRICT-9-conjugated Sepharose 4B was mixed with the cell lysates or STAT3 recombinant protein (Abnova, Taipei, Taiwan) at 4°C for 24 h and washed three times with TBST. The bound proteins were diluted with SDS loading buffer and resolved by SDS-PAGE followed by immunoblotting with STAT3 antibodies (1:1000 dilution, Santa Cruz Biotechnology).