American Society of Hematology

Figure 2

$Figure 2. Serine pSTAT3 translocates to the nucleus. (A) Cytoplasmic (Cyt.) and nuclear (Nuc.) fractions were extracted from CLL cells of patients 2, 11, 12, and 13, as described in “Isolation of nuclear and cytoplasmic extracts,” and analyzed by Western immunoblotting using antiserine pSTAT3 and anti-STAT3 antibodies. Adequate fractionation of cytoplasmic and nuclear extracts was confirmed using anti-S6 ribosomal protein and antilamin B1 antibodies. (B) Confocal microscopic images of freshly isolated CLL cells (CLL 14 and 15) that were cytospun and fixed on glass slides, as described in “Confocal microscopy.” As shown (original magnification × 400), the slides were stained with Alexa Fluor 488–mouse antiphosphoserine (serine 727)-STAT3 antibodies (red dots; bottom left corner) and the nuclear stain TOPRO3 (blue; top right corner). Serine pSTAT3 was detected in the nucleus of CLL cells (red dots overlapping blue; bottom right corner). (C) Using a different approach, the slides were stained with antiphosphoserine (serine 727)-STAT3 Alexa Fluor 488–mouse antibodies (green dots) and ribosomal protein S6, followed by phycoerythrin-conjugated rabbit anti–mouse antibodies (red dots). Serine pSTAT3 was detected in the nucleus (green dots) and the cytoplasm (yellow dots represent colocalization of ribosomal protein S6 and serine pSTAT3; X-1000). Vertical lines indicate realignment of the same gel's image. Images were acquired using an Olympus Flowview FV500/X81 system using Flowview software and a 60× oil immersion lens, and cropped and revised using Microsoft Office PowerPoint 2003.$

Serine pSTAT3 translocates to the nucleus. (A) Cytoplasmic (Cyt.) and nuclear (Nuc.) fractions were extracted from CLL cells of patients 2, 11, 12, and 13, as described in “Isolation of nuclear and cytoplasmic extracts,” and analyzed by Western immunoblotting using antiserine pSTAT3 and anti-STAT3 antibodies. Adequate fractionation of cytoplasmic and nuclear extracts was confirmed using anti-S6 ribosomal protein and antilamin B1 antibodies. (B) Confocal microscopic images of freshly isolated CLL cells (CLL 14 and 15) that were cytospun and fixed on glass slides, as described in “Confocal microscopy.” As shown (original magnification × 400), the slides were stained with Alexa Fluor 488–mouse antiphosphoserine (serine 727)-STAT3 antibodies (red dots; bottom left corner) and the nuclear stain TOPRO3 (blue; top right corner). Serine pSTAT3 was detected in the nucleus of CLL cells (red dots overlapping blue; bottom right corner). (C) Using a different approach, the slides were stained with antiphosphoserine (serine 727)-STAT3 Alexa Fluor 488–mouse antibodies (green dots) and ribosomal protein S6, followed by phycoerythrin-conjugated rabbit anti–mouse antibodies (red dots). Serine pSTAT3 was detected in the nucleus (green dots) and the cytoplasm (yellow dots represent colocalization of ribosomal protein S6 and serine pSTAT3; X-1000). Vertical lines indicate realignment of the same gel's image. Images were acquired using an Olympus Flowview FV500/X81 system using Flowview software and a 60× oil immersion lens, and cropped and revised using Microsoft Office PowerPoint 2003.

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