Figure 2.
Figure 2. Coclustering with CD3ζ and nuclear translocation of Homer-3 in response to TCR stimulation in T cells. (A) Jurkat cells were incubated for 2 minutes at 37°C on glass slides coated with poly-l-lysine only or poly-l-lysine and anti-CD3 antibody (OKT3). After fixation, the cells were stained with anti-CD3ζ antibody and anti–Homer-3 antibody followed by secondary antibodies-Alexa Fluor 594 (red) or 488 (green). After incubation on the glass slides coated with OKT3, Homer-3 (green) coclustered with CD3ζ (red). (B) Jurkat cells were incubated for 10 minutes at 37°C on glass slides coated with poly-l-lysine only or poly-l-lysine and OKT3. After fixation, the cells were stained with anti–Homer-3 antibody followed by secondary antibody-Alexa Fluor 488 (green), phalloidin-Alexa Fluor 594 (red), and DAPI (blue) for nuclear staining. Homer-3 (green) was distributed in the cytoplasmic region after incubation on the glass slides coated with poly-l-lysine only. After incubation on the glass slides coated with OKT3, F-actin (red) was rearranged into circumferential rings and Homer-3 was translocated into the nucleus (blue). The nuclear translocation of Homer-3 was also observed in Jurkat cells stimulated with PMA in solution for 10 minutes. (C) Jurkat cells were incubated for 10 minutes as in panel B. After fixation, the cells were stained with anti–Homer-3 antibody followed by secondary antibody-Alexa Fluor 488 (green) and propidium iodide (red) for nuclear staining. The cells were observed with confocal laser scanning microscopy, which confirmed the nuclear translocation of Homer-3 following TCR stimulation. (D) Primary CD4+ T cells were isolated from mouse spleen and stimulated on antimouse CD3 antibody–coated glass slides. While Homer-3 was distributed in the cytoplasmic region of the cells incubated on the glass slides coated with poly-l-lysine only, the translocation into the nucleus was observed after stimulation with anti-CD3 antibody. Representative images were presented from experiments performed at least 3 times. Bars, 10 μm.

Coclustering with CD3ζ and nuclear translocation of Homer-3 in response to TCR stimulation in T cells. (A) Jurkat cells were incubated for 2 minutes at 37°C on glass slides coated with poly-l-lysine only or poly-l-lysine and anti-CD3 antibody (OKT3). After fixation, the cells were stained with anti-CD3ζ antibody and anti–Homer-3 antibody followed by secondary antibodies-Alexa Fluor 594 (red) or 488 (green). After incubation on the glass slides coated with OKT3, Homer-3 (green) coclustered with CD3ζ (red). (B) Jurkat cells were incubated for 10 minutes at 37°C on glass slides coated with poly-l-lysine only or poly-l-lysine and OKT3. After fixation, the cells were stained with anti–Homer-3 antibody followed by secondary antibody-Alexa Fluor 488 (green), phalloidin-Alexa Fluor 594 (red), and DAPI (blue) for nuclear staining. Homer-3 (green) was distributed in the cytoplasmic region after incubation on the glass slides coated with poly-l-lysine only. After incubation on the glass slides coated with OKT3, F-actin (red) was rearranged into circumferential rings and Homer-3 was translocated into the nucleus (blue). The nuclear translocation of Homer-3 was also observed in Jurkat cells stimulated with PMA in solution for 10 minutes. (C) Jurkat cells were incubated for 10 minutes as in panel B. After fixation, the cells were stained with anti–Homer-3 antibody followed by secondary antibody-Alexa Fluor 488 (green) and propidium iodide (red) for nuclear staining. The cells were observed with confocal laser scanning microscopy, which confirmed the nuclear translocation of Homer-3 following TCR stimulation. (D) Primary CD4+ T cells were isolated from mouse spleen and stimulated on antimouse CD3 antibody–coated glass slides. While Homer-3 was distributed in the cytoplasmic region of the cells incubated on the glass slides coated with poly-l-lysine only, the translocation into the nucleus was observed after stimulation with anti-CD3 antibody. Representative images were presented from experiments performed at least 3 times. Bars, 10 μm.

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