Auxin-induced degradation of Mcm4-aid. (A) Amounts of Mcm4-aid and TIR1 in the presence or absence of auxin. HM1905 mcm4-aid Pnmt41-AtTIR1 cells were cultured at 25°C in the presence (lanes 5-8) or absence (lanes 1-4) of auxin (0.5 mM) for indicated periods and proteins in whole-cell extracts were separated in 7.5% polyacrylamide gel and analyzed by immunoblotting with anti-Mcm4, myc (TIR1), and TAT1 (tubulin) antibodies. (B) The amount of Mcm4-aid after depletion was compared to that in the 0 h sample diluted with SDS sample buffer to 50%, 25%, and 12.5%. (C) Flow cytometry analysis of mcm4-aid Pnmt41-AtTIR1 cells. HM1905 mcm4-aid Pnmt41-AtTIR1 cells were cultured at 25°C and collected every 1 h after addition of auxin (0.5 mM), and then the DNA contents of the cells were analyzed. Positions of 1C and 2C DNA contents are indicated. (D) Growth on auxin plates. Log-phase cultures of wild type, mcm4-aid without TIR1 (HM1909) and mcm4-aid Pnmt41-AtTIR1 (HM1905) were serially diluted and spotted onto EMM plates with or without auxin (0.5 mM) and incubated at 25°C.
Kanke et al. BMC Cell Biology 2011 12:8 doi:10.1186/1471-2121-12-8