|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. (A) In situ extraction experiments of HMGA1a-GFP. (a) Permeabilized cells expressing HMGA1a-GFP incubated in low salt extraction buffer retained nuclear fluorescence. Incubation in buffer containing 350 mM salt resulted in a loss of nuclear fluorescence (e) as well as after treatment with DNase I (j). Localization of the non-snRNP splicing factor sc35 was used as a control for the extraction experiments (c,g,l). Note that the distribution of sc35 and HMGA1a-GFP does not overlap (d). DNA was counterstained with Hoechst 33258 (b-k). Overlays are shown in (d,h,m). Cells were monitored using a Zeiss Axiophot equipped with a Pixera Digital Imaging System. Bars represent 10µm. (B) Extractability of HMGA1a-GFP as analyzed in western blot experiments. Cells transfected with HMGA1a-GFP were extracted with PBS (140 mM salt) or PBS containing 350mM salt, respectively. Solubilized proteins were precipitated and submitted to western blot analyses. Stripped blots were reprobed with antibodies directed to HMGA1 proteins, HMGN proteins or actin. HMGA1a-GFP was detected with an antibody directed against GFP. Note that comparable with endogenous HMGA1 (b) and HMGN (c) proteins, HMGA1a-GFP (a) are extracted in 350 mM salt. Control cells expressing GFP were treated like described above. Solubility of GFP is independent of salt treatment (e). Detection of cellular actin was used to show equal loading of extracted proteins (d,f).
|
