Figure 3.
OxLDL-CD36 signaling promotes SFK cysteine sulfenylation. (A) oxLDL promotes cysteine modification of SFK in platelets. Washed human platelets (3 × 108/mL) were stimulated with buffer or 50 µg/mL oxLDL up to 60 minutes or with 500 µM H2O2 as a positive control for 15 minutes. Platelets were lysed, and unmodified cysteine thiols were alkylated with 5 µM of 5-IAF; oxidized thiols are unable to be alkylated by 5-IAF. SFK were immunoprecipitated and washed, and 5-IAF was detected by an anti-fluorescein antibody. Total SFK were detected by an anti-Src antibody. (B) oxLDL promotes SFK cysteine sulfenylation over time. (C) SFK cysteine sulfenylation by oxLDL is CD36 dependent. (D) H2O2 is the ROS downstream of CD36 that sulfenylates SFK. Washed human platelets (3 × 108/mL) were pretreated with 1 mM BTD-alkyne (B) in the presence of 1 µg/mL CD36-blocking FA6-152 (FA6) or control IgG antibody (C), or with 1000 U/mL of denatured or functional PEG-catalase (PEG-Cat) (D). The platelets were then stimulated with 50 µg/mL oxLDL up to 60 minutes (B-D), or with 500 µM H2O2 as a positive control for 15 minutes (B). Levels of SFK immunoprecipitated were assessed by UV-dependent stain-free imaging of the Bio-Rad TGX gels. (E) oxLDL/CD36 signaling promotes activation of SFK over time. (F) SFK activation by CD36 is H2O2 dependent. Washed human platelets (3 × 108/mL) were stimulated with 50 µg/mL oxLDL up to 60 minutes or with 500 µM H2O2 as a positive control (E) or with 50 µg/mL oxLDL following pretreatment with 1000 U/mL functional or denatured PEG-catalase (PEG-Cat) (F) for 15 minutes. The cells were lysed with radioimmunoprecipitation assay buffer, and phosphorylated SFK (Y416) were detected by immunoblotting. Total SFK was detected by an anti-Src antibody. P values were determined by 1-way analysis of variance with Dunnett’s posthoc analysis (A-C,E) and by 1-way analysis of variance with Tukey’s post hoc analysis (D,F). N = 3 separate donors (A-F). *P < .05 compared with buffer treatment (A-C,E). *P < .05 with their respective comparisons (D,F). Data are expressed as mean ± SEM.

OxLDL-CD36 signaling promotes SFK cysteine sulfenylation. (A) oxLDL promotes cysteine modification of SFK in platelets. Washed human platelets (3 × 108/mL) were stimulated with buffer or 50 µg/mL oxLDL up to 60 minutes or with 500 µM H2O2 as a positive control for 15 minutes. Platelets were lysed, and unmodified cysteine thiols were alkylated with 5 µM of 5-IAF; oxidized thiols are unable to be alkylated by 5-IAF. SFK were immunoprecipitated and washed, and 5-IAF was detected by an anti-fluorescein antibody. Total SFK were detected by an anti-Src antibody. (B) oxLDL promotes SFK cysteine sulfenylation over time. (C) SFK cysteine sulfenylation by oxLDL is CD36 dependent. (D) H2O2 is the ROS downstream of CD36 that sulfenylates SFK. Washed human platelets (3 × 108/mL) were pretreated with 1 mM BTD-alkyne (B) in the presence of 1 µg/mL CD36-blocking FA6-152 (FA6) or control IgG antibody (C), or with 1000 U/mL of denatured or functional PEG-catalase (PEG-Cat) (D). The platelets were then stimulated with 50 µg/mL oxLDL up to 60 minutes (B-D), or with 500 µM H2O2 as a positive control for 15 minutes (B). Levels of SFK immunoprecipitated were assessed by UV-dependent stain-free imaging of the Bio-Rad TGX gels. (E) oxLDL/CD36 signaling promotes activation of SFK over time. (F) SFK activation by CD36 is H2O2 dependent. Washed human platelets (3 × 108/mL) were stimulated with 50 µg/mL oxLDL up to 60 minutes or with 500 µM H2O2 as a positive control (E) or with 50 µg/mL oxLDL following pretreatment with 1000 U/mL functional or denatured PEG-catalase (PEG-Cat) (F) for 15 minutes. The cells were lysed with radioimmunoprecipitation assay buffer, and phosphorylated SFK (Y416) were detected by immunoblotting. Total SFK was detected by an anti-Src antibody. P values were determined by 1-way analysis of variance with Dunnett’s posthoc analysis (A-C,E) and by 1-way analysis of variance with Tukey’s post hoc analysis (D,F). N = 3 separate donors (A-F). *P < .05 compared with buffer treatment (A-C,E). *P < .05 with their respective comparisons (D,F). Data are expressed as mean ± SEM.

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