American Society of Hematology

Figure 3

$Figure 3. The entire AnxA2-S100A10 complex is involved in cAMP-dependent VWF release from HUVECs. HUVECs were transfected with siRNA specific for AnxA2 or nontargeting siRNA (ct RNAi). (A) Forty-eight hours later, cells were subjected to a second transfection using control siRNA, AnxA2 siRNA, or AnxA2 siRNA plus an siRNA-insensitive AnxA2 expression vector (AII-Rr). PNSs were prepared and analyzed by immunoblotting using rabbit polyclonal anti-AnxA2 antibodies for the detection of the total (endogenous and ectopically expressed) AnxA2 (total) and mouse monoclonal anti-AnxA2 antibodies (clone H28) for the specific detection of only ectopically expressed AnxA2 (ect.; see supplemental Methods). Probing with monoclonal α-tubulin antibodies was included as a loading control. (B) HUVECs transiently transfected with AnxA2 siRNA plus rescue plasmid (AII-Rr) were stained with (left) anti-AnxA2 antibodies specifically labeling the ectopically expressed AnxA2 (clone H28) and (right) anti-S100A10 antibodies directly coupled to Alexa Fluor 594. Nuclei were labeled with DAPI (blue). Scale bars represent 10 µm. Note the efficient restoration of AnxA2 and endogenous S100A10 levels in cells expressing the AnxA2 rescue construct (AII-Rr). (C) Cells transfected with siRNA (AnxA2 or nontargeting siRNA) or siRNA plus rescue plasmid (AII-Rr) were subjected to secretagogue-free and forskolin/IBMX-containing stimulation media for 20 min. Subsequently, the amount of released VWF was analyzed by enzyme-linked immunosorbent assay (ELISA). The effect on secretion was quantified in sets of 13 independent experiments, and 1-way ANOVA with Bonferroni test was performed to evaluate statistical significance. Bars represent mean ± SEM. (D) Forty-eight hours after the first siRNA transfection, HUVECs were subjected to a second round of transfection using either AnxA2 siRNA, AnxA2 siRNA plus a ubiquitinylation-resistant S100A10 mutant construct (YFP-S100A10-CΔ6), or control siRNA. Following another 48 h, PNS fractions were prepared and subjected to immunoblotting using rabbit polyclonal anti-AnxA2 and mouse monoclonal anti-S100A10 antibodies. Probing with monoclonal α-tubulin antibodies served as an internal loading control. Note the stability of the S100A10 mutant in cells depleted of AnxA2. (E) Transfections were carried out as described in D, and the amount of VWF released in response to forskolin was quantified by ELISA in sets of 3 independent experiments. One-way ANOVA with Bonferroni test was used for the evaluation of statistical significance. Bars represent mean ± SEM.$

The entire AnxA2-S100A10 complex is involved in cAMP-dependent VWF release from HUVECs. HUVECs were transfected with siRNA specific for AnxA2 or nontargeting siRNA (ct RNAi). (A) Forty-eight hours later, cells were subjected to a second transfection using control siRNA, AnxA2 siRNA, or AnxA2 siRNA plus an siRNA-insensitive AnxA2 expression vector (AII-Rr). PNSs were prepared and analyzed by immunoblotting using rabbit polyclonal anti-AnxA2 antibodies for the detection of the total (endogenous and ectopically expressed) AnxA2 (total) and mouse monoclonal anti-AnxA2 antibodies (clone H28) for the specific detection of only ectopically expressed AnxA2 (ect.; see supplemental Methods). Probing with monoclonal α-tubulin antibodies was included as a loading control. (B) HUVECs transiently transfected with AnxA2 siRNA plus rescue plasmid (AII-Rr) were stained with (left) anti-AnxA2 antibodies specifically labeling the ectopically expressed AnxA2 (clone H28) and (right) anti-S100A10 antibodies directly coupled to Alexa Fluor 594. Nuclei were labeled with DAPI (blue). Scale bars represent 10 µm. Note the efficient restoration of AnxA2 and endogenous S100A10 levels in cells expressing the AnxA2 rescue construct (AII-Rr). (C) Cells transfected with siRNA (AnxA2 or nontargeting siRNA) or siRNA plus rescue plasmid (AII-Rr) were subjected to secretagogue-free and forskolin/IBMX-containing stimulation media for 20 min. Subsequently, the amount of released VWF was analyzed by enzyme-linked immunosorbent assay (ELISA). The effect on secretion was quantified in sets of 13 independent experiments, and 1-way ANOVA with Bonferroni test was performed to evaluate statistical significance. Bars represent mean ± SEM. (D) Forty-eight hours after the first siRNA transfection, HUVECs were subjected to a second round of transfection using either AnxA2 siRNA, AnxA2 siRNA plus a ubiquitinylation-resistant S100A10 mutant construct (YFP-S100A10-CΔ6), or control siRNA. Following another 48 h, PNS fractions were prepared and subjected to immunoblotting using rabbit polyclonal anti-AnxA2 and mouse monoclonal anti-S100A10 antibodies. Probing with monoclonal α-tubulin antibodies served as an internal loading control. Note the stability of the S100A10 mutant in cells depleted of AnxA2. (E) Transfections were carried out as described in D, and the amount of VWF released in response to forskolin was quantified by ELISA in sets of 3 independent experiments. One-way ANOVA with Bonferroni test was used for the evaluation of statistical significance. Bars represent mean ± SEM.

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