American Society of Hematology

Figure 6

$Figure 6. IBtkγ S87/90A prevents the Btk translocation to the plasma membrane on BCR triggering. (A-C) IBtkγ prevents the Btk translocation to the plasma membrane on BCR triggering in a PI3K-independent manner. DeFew cells stably expressing empty vector (A), FLAG-IBtkγ (B), or FLAG-IBtkγ S87/90A (C) were stimulated with anti-IgM for 5 minutes in the presence or absence of the PI3K inhibitor LY294002 (25μM); then, cells were fixed and stained with either anti-Btk Ab followed by anti–rabbit Alexa 568 to detect endogenous Btk, and FITC-conjugated anti-FLAG Ab to detected FLAG-IBtkγ. Nuclei were stained with DAPI. Images shown are the distribution of transfected IBtkγ (green), endogenous Btk (red), and nuclei (blue) were along a single focal plane detected by confocal microscopy. Arrowheads indicate the punctuate aggregation of Btk at the plasma membrane. Bar = 10 μm. A representative experiment of 2 independent experiments is shown. (D) Immunoblotting analysis of the Btk cellular distribution. DeFew cells stably expressing FLAG-IBtkγ or control empty plasmid were stimulated with anti-IgM for 5 and 20 minutes, and cell lysates were subjected to subcellular fractionation. The Btk expression levels in the cytosolic and membrane fractions (10 μg) were analyzed by Western blotting with anti-Btk antibodies; RACK1 and transferrin receptor (TrR) were analyzed with the relative antibodies as markers of the cytosol and membrane, respectively. Densitometry of Btk expression levels in the cytosolic and membrane fractions was performed by the use of Scion Image; for each point, the Btk signal was normalized to the relative markers RACK1 and TrR. A representative experiment of 2 independent experiments is shown. (E) Association of Btk and BLNK in DeFew cells. DeFew cells stably expressing empty vector (A), FLAG-IBtkγ (B), or FLAG-IBtkγ S87/90A (C) were stimulated with anti-IgM for 5 minutes or left unstimulated. Cell lysates (1 mg) were immunoprecipitated with anti-BLNK Ab and analyzed by 4%-12% NuPAGE followed by immunoblotting with anti-Btk, anti-Syk, and anti-BLNK antibodies. A representative experiment of 2 independent experiments is shown.$

IBtkγ S87/90A prevents the Btk translocation to the plasma membrane on BCR triggering. (A-C) IBtkγ prevents the Btk translocation to the plasma membrane on BCR triggering in a PI3K-independent manner. DeFew cells stably expressing empty vector (A), FLAG-IBtkγ (B), or FLAG-IBtkγ S87/90A (C) were stimulated with anti-IgM for 5 minutes in the presence or absence of the PI3K inhibitor LY294002 (25μM); then, cells were fixed and stained with either anti-Btk Ab followed by anti–rabbit Alexa 568 to detect endogenous Btk, and FITC-conjugated anti-FLAG Ab to detected FLAG-IBtkγ. Nuclei were stained with DAPI. Images shown are the distribution of transfected IBtkγ (green), endogenous Btk (red), and nuclei (blue) were along a single focal plane detected by confocal microscopy. Arrowheads indicate the punctuate aggregation of Btk at the plasma membrane. Bar = 10 μm. A representative experiment of 2 independent experiments is shown. (D) Immunoblotting analysis of the Btk cellular distribution. DeFew cells stably expressing FLAG-IBtkγ or control empty plasmid were stimulated with anti-IgM for 5 and 20 minutes, and cell lysates were subjected to subcellular fractionation. The Btk expression levels in the cytosolic and membrane fractions (10 μg) were analyzed by Western blotting with anti-Btk antibodies; RACK1 and transferrin receptor (TrR) were analyzed with the relative antibodies as markers of the cytosol and membrane, respectively. Densitometry of Btk expression levels in the cytosolic and membrane fractions was performed by the use of Scion Image; for each point, the Btk signal was normalized to the relative markers RACK1 and TrR. A representative experiment of 2 independent experiments is shown. (E) Association of Btk and BLNK in DeFew cells. DeFew cells stably expressing empty vector (A), FLAG-IBtkγ (B), or FLAG-IBtkγ S87/90A (C) were stimulated with anti-IgM for 5 minutes or left unstimulated. Cell lysates (1 mg) were immunoprecipitated with anti-BLNK Ab and analyzed by 4%-12% NuPAGE followed by immunoblotting with anti-Btk, anti-Syk, and anti-BLNK antibodies. A representative experiment of 2 independent experiments is shown.

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