Page 119 - 2019_01-Haematologica-web

2-Bromopalmitate promotes APL differentiation
B
C
A
Figure 6. Identificaiton of 2BP binding sites within RARα. (A) Structure schematic diagram of RARα protein. AF: activation function domain; DBD: DNA binding domain; LBD: ligand binding domain. (B-C) MS/MS analysis of the Cys105-(B) or Cys174- containing(C) tryptic peptide for recombinant RARα incubated without (top) and with (bottom) 2BP for 30 mins. (D) Recombinant wild-type (WT) RARα and the Cys105/174 double mutants(DM) were incubated with biotin-2BP for 30 mins, fol- lowed by blotting with anti-RARα antibody. The membrane stained with Ponceau S is shown at the bottom. (E) NB4 cells were infected with shRNA specifically against RARα to deplete endogeneous RARα and then infected with plasmids encompassing WT or DM RARα. These cells were treated with 5 mM 2BP and/or 10-8M ATRA for 3 days and CD11b-positive cells were counted by flow cytometry. *P<0.05 against ATRA-treated group. (F) NB4 cells expressing DM RARα were incubated with 5 mM of 2BP and/or 10-8M ATRA for 48 hours and RARα protein was detected by western blot with β-actin as the loading control.
haematologica | 2019; 104(1)
109
DEF