Skip to main content
. 2011 Oct 3;6(10):e25743. doi: 10.1371/journal.pone.0025743

Figure 3. Characterization of doxycycline inducible coilin phosphomutant cell lines.

Figure 3

(A and B) Stable cell lines expressing GFP-coilin of GFP-coilin phosphomutant proteins. Non-induced (−) and 24 h doxycycline induced (1 µg/mL, +) cell lysates of GFP-coilin (WT) and GFP-coilin phosphomutants (T122E, ON, S489D, S271/272D or OFF) were subjected to SDS-PAGE, followed by western transfer. The blots were probed with mouse monoclonal anti-GFP antibodies for specific detection of the GFP-tagged coilin proteins (upper panel). The blots were re-probed with rabbit polyclonal anti-coilin antibodies to detect both endogenous coilin and the GFP-tagged WT and coilin phosphomutants (lower panel). Note that S489D and OFF expression generates an approximately 42 kDa degradation product. (C) Transiently transfected GFP-coilin S489D and GFP-coilin OFF phosphomutant proteins do not have a specific degradation product. HeLa cells were transiently transfected with GFP-coilin S489D and GFP-coilin OFF DNA constructs for 24 h followed by lysate generation, SDS-PAGE and western transfer. The blot was probed with antibodies to GFP. (D) Full length GFP-coilin S489D can be detected after doxycycline induction by immunoprecipitation. Doxycycline (0.33 or 1 µg/mL) induced and non-induced GFP-coilin-S489D stable cell extracts were immunoprecipitated with anti-GFP antibodies. The western blot was probed with anti-GFP antibodies for the detection of the GFP-coilin-S489D fragment (upper panel). The same blot was probed with anti-coilin antibodies for endogenous coilin and full length GFP-coilin-S489D protein detection (lower panel). IgG(H) denotes the immunoglobulin heavy chain.