Journal: PLoS ONE

Article Title: BCR-ABL Affects STAT5A and STAT5B Differentially

doi: 10.1371/journal.pone.0097243

Figure Lengend Snippet: (A) TonB cells cultured in the presence of IL-3 or doxycycline were spun down on glass slides, fixed by ice-cold methanol and stained sequentially with anti-STAT5B, anti-rabbit-FITC and DAPI. Intracellular localization was analyzed using confocal microscopy. Nuclear localization was confirmed by a DNA-specific DAPI counterstain. The lower panel shows antibody (with and without enhancement) and DAPI control. (B) TonB cells expressing STAT5A-eGFP were cultured in the presence of IL-3, BCR-ABL or BCR-ABL plus imatinib mesylate (BCR-ABL+IM) and live-cell analysis was performed by confocal microscopy (upper panel). The middle panel shows STAT5A-eGFP in BCR-ABL-expressing TonB cells after removal of imatinib mesylate (−IM) for the indicated periods of time. The lower panel shows empty vector control (eGFP). (C) TonB-STAT5A-eGFP cells treated as in , middle panel, were fixed 45 minutes after imatinib-removal, stained with anti-CD131-PE antibody (IL-3/IL-5/GM-CSF-receptor common β-chain) and DAPI before analysis by confocal microscopy.

Article Snippet: Cells were blocked in PBS supplemented with 10% [v/v] FCS (Biochrome, Berlin, Germany) for 1 hour and stained with PE Rat anti-mouse CD131 (559920) from BD Pharmingen and DAPI (0.5 µg/mL) for 2 hours.

Techniques: Cell Culture, Staining, Confocal Microscopy, Expressing, Plasmid Preparation