Figure 8.
Figure 8. Kinesin-1–dependent transport machinery formation requires the activation of PI3K. (A) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO (control), 100 µM PI3K inhibitor LY-294002, or 20 mM intracellular calcium chelator BAPTA, and then stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed and incubated with anti-CD63 antibody before flow cytometry analysis (left), and expression was quantified (ΔMFI CD63+; right). (B) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO, LY-294002, or BAPTA. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min and then lysed. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-Rab27 antibody or with isotype control antibody. The immunoblots were analyzed using anti-Kif5b, anti-Slp3, and anti-Rab27 antibodies. Data are representative of three independent experiments. (C, left) IgE-sensitized BMMCs were preincubated in medium containing DMSO, 100 µM LY-294002, or 20 mM BAPTA. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed, permeabilized, and stained with anti-STX3 and antitubulin antibodies. (Right) More than 100 cells were counted per setting. (D) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO (control) or 20 µM nocodazole and then stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed and incubated with anti-CD63 antibody before flow cytometry analysis (left), and expression was quantified (ΔMFI CD63+; right). (E) IgE-sensitized BMMCs were preincubated in medium containing DMSO or nocodazole. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed, permeabilized, and stained with anti-STX3 and antitubulin antibodies (left). Bars, 2 µm. (Right) More than 100 cells were counted per setting. Statistical analyses were performed using unpaired t tests. ***, P < 0.0001. (F) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO or 20 µM nocodazole. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min before lysis. Cell lysates were immunoprecipitated with a polyclonal anti-Rab27 antibody or with isotype control antibody. The immunoblots were analyzed using anti-Kif5b, anti-Slp3, and anti-Rab27 antibodies. Data are representative of three independent experiments. Error bars represent mean ± SD. MFI, mean fluorescence intensity.

Kinesin-1–dependent transport machinery formation requires the activation of PI3K. (A) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO (control), 100 µM PI3K inhibitor LY-294002, or 20 mM intracellular calcium chelator BAPTA, and then stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed and incubated with anti-CD63 antibody before flow cytometry analysis (left), and expression was quantified (ΔMFI CD63+; right). (B) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO, LY-294002, or BAPTA. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min and then lysed. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-Rab27 antibody or with isotype control antibody. The immunoblots were analyzed using anti-Kif5b, anti-Slp3, and anti-Rab27 antibodies. Data are representative of three independent experiments. (C, left) IgE-sensitized BMMCs were preincubated in medium containing DMSO, 100 µM LY-294002, or 20 mM BAPTA. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed, permeabilized, and stained with anti-STX3 and antitubulin antibodies. (Right) More than 100 cells were counted per setting. (D) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO (control) or 20 µM nocodazole and then stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed and incubated with anti-CD63 antibody before flow cytometry analysis (left), and expression was quantified (ΔMFI CD63+; right). (E) IgE-sensitized BMMCs were preincubated in medium containing DMSO or nocodazole. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed, permeabilized, and stained with anti-STX3 and antitubulin antibodies (left). Bars, 2 µm. (Right) More than 100 cells were counted per setting. Statistical analyses were performed using unpaired t tests. ***, P < 0.0001. (F) IgE-sensitized BMMCs were preincubated in normal medium containing DMSO or 20 µM nocodazole. BMMCs were stimulated with 20 ng/ml DNP-HSA for 10 min before lysis. Cell lysates were immunoprecipitated with a polyclonal anti-Rab27 antibody or with isotype control antibody. The immunoblots were analyzed using anti-Kif5b, anti-Slp3, and anti-Rab27 antibodies. Data are representative of three independent experiments. Error bars represent mean ± SD. MFI, mean fluorescence intensity.

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