Figure 4
Figure 4. Lack of enzymatic activity of CyPA prevents phosphorylation of STIM1 and reduces cell adhesion on fibrinogen. Resting or thrombin-activated (0.1 U/mL) platelets were lysed, immunoprecipitated with the appropriate Abs, and immunoblotted with the indicated Abs detecting STIM1, Orai1, and CyPA, respectively. (A) Immunoprecipitation of whole-cell lysates of Cypa+/+ and Cypa−/− platelets with STIM1 Ab, followed by the detection of CyPA by Western blot analysis. Reproving of blots after stripping was performed with STIM1 Ab serving as a loading control. (B) Thrombin (0.1 U/mL)–stimulated Cypa+/+, Cypa−/−, and CsA-treated Cypa+/+ platelets (1 and 5 minutes) were lysed and immunoprecipitated with anti-phosphotyrosine Ab (4G10). Immunoprecipitates were analyzed by Western blot using STIM1 Ab showing rapid phosphorylation of STIM1 only in Cypa+/+ platelets. (C) Whole-cell lysates of thrombin-stimulated platelets were immunoprecipitated with Orai1 Ab, followed by Western blot analysis with STIM1 Ab showing colocalization of STIM1 and Orai1 independent of STIM1 phosphorylation. STIM1/Orai1 complex formation was also observed on CsA treatment. (D) Thrombin (0.1 U/mL)–stimulated Cypa+/+, Cypa−/−, and CsA-treated Cypa+/+ platelets (1 and 5 minutes) were lysed and immunoprecipitated with anti-phosphotyrosine Ab (4G10). Immunoprecipitates were analyzed by Western blot using ERK1/2 Ab showing comparable phosphorylation of ERK1/2 in control and Cypa−/−- and CsA-treated platelets. (E) Immunoprecipitation of platelet lysates of Cypa+/+ and Cypa−/− platelets with SERCA2b Ab, followed by the detection of CyPA by Western blot analysis. To assess that a similar amount of proteins were loaded, membranes were reproved with SERCA2b Ab serving as a loading control. (F) After thrombin stimulation, platelets were lysed and immunoprecipitated with STIM1 Ab, followed by Western blot analysis with SERCA2b Ab showing colocalization of STIM1 and SERCA2b in murine platelets. STIM1/SERCA2b complex formation was also observed in CyPA-deficient platelets and on CsA treatment. (G) A5-CHO cells expressing GFP, STIM1-GFP, or STIM1S575A/S608A/S621A-GFP were allowed to adhere and spread on human fibrinogen (1 mg/mL) for 60 minutes. STIM1 was detected by the fluorescence properties of GFP. Scale bar indicates 20 μm. (H) Statistical analysis of adhesive cells per visual field. The bar graph depicts mean values ± SD (n = 10). ***P < .001.

Lack of enzymatic activity of CyPA prevents phosphorylation of STIM1 and reduces cell adhesion on fibrinogen. Resting or thrombin-activated (0.1 U/mL) platelets were lysed, immunoprecipitated with the appropriate Abs, and immunoblotted with the indicated Abs detecting STIM1, Orai1, and CyPA, respectively. (A) Immunoprecipitation of whole-cell lysates of Cypa+/+ and Cypa−/− platelets with STIM1 Ab, followed by the detection of CyPA by Western blot analysis. Reproving of blots after stripping was performed with STIM1 Ab serving as a loading control. (B) Thrombin (0.1 U/mL)–stimulated Cypa+/+, Cypa−/−, and CsA-treated Cypa+/+ platelets (1 and 5 minutes) were lysed and immunoprecipitated with anti-phosphotyrosine Ab (4G10). Immunoprecipitates were analyzed by Western blot using STIM1 Ab showing rapid phosphorylation of STIM1 only in Cypa+/+ platelets. (C) Whole-cell lysates of thrombin-stimulated platelets were immunoprecipitated with Orai1 Ab, followed by Western blot analysis with STIM1 Ab showing colocalization of STIM1 and Orai1 independent of STIM1 phosphorylation. STIM1/Orai1 complex formation was also observed on CsA treatment. (D) Thrombin (0.1 U/mL)–stimulated Cypa+/+, Cypa−/−, and CsA-treated Cypa+/+ platelets (1 and 5 minutes) were lysed and immunoprecipitated with anti-phosphotyrosine Ab (4G10). Immunoprecipitates were analyzed by Western blot using ERK1/2 Ab showing comparable phosphorylation of ERK1/2 in control and Cypa−/−- and CsA-treated platelets. (E) Immunoprecipitation of platelet lysates of Cypa+/+ and Cypa−/− platelets with SERCA2b Ab, followed by the detection of CyPA by Western blot analysis. To assess that a similar amount of proteins were loaded, membranes were reproved with SERCA2b Ab serving as a loading control. (F) After thrombin stimulation, platelets were lysed and immunoprecipitated with STIM1 Ab, followed by Western blot analysis with SERCA2b Ab showing colocalization of STIM1 and SERCA2b in murine platelets. STIM1/SERCA2b complex formation was also observed in CyPA-deficient platelets and on CsA treatment. (G) A5-CHO cells expressing GFP, STIM1-GFP, or STIM1S575A/S608A/S621A-GFP were allowed to adhere and spread on human fibrinogen (1 mg/mL) for 60 minutes. STIM1 was detected by the fluorescence properties of GFP. Scale bar indicates 20 μm. (H) Statistical analysis of adhesive cells per visual field. The bar graph depicts mean values ± SD (n = 10). ***P < .001.

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