Journal: bioRxiv

Article Title: aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete β-arrestin-2 mediated SphK1-S1PR1-Akt signaling axis

doi: 10.1101/2021.03.27.437291

Figure Lengend Snippet: (A) EA.hy926 cells were stimulate with or without aPC for 180 min, phospho-SphK1 was detected by immunoblotting. Data (mean ± S.D., n = 3) were analyzed by Student’s t-test. (B) EA.hy926 cells were stimulated with or without 20 nM aPC for 30 min, cells were lysed and subcellular fractionation performed. SphK1, the cytoplasmic control (GAPDH) and the membrane control (Na + /K + ATPase) were detected by immunoblotting. Data (mean ± S.D., n = 3) was analyzed by Student’s t-test. (C) EA.hy926 cells were pretreated with or without 100 nM PF-543 for 1 h, and then treated with 20 nM of aPC for 15 min. Cells were lysed and SphK1 activity assessed by using the Echelon SphK1 activity assay. Data (mean ± S.D., n = 3) was analyzed by Student’s t-test. (D) EA.hy926 cells were pretreated with or without 100 nM PF-543 for 1 h and stimulated with or without 20 nM aPC for various times, phospho-Akt-S473 was detected by immunoblotting. Data (mean ± S.D., n = 3) was analyzed by two-way ANOVA. (E) HUVECs were treated and analyzed as described in D. ****, P <0.0001, ***, P <0.001, **, P <0.01, and *, P <0.05.

Article Snippet: Serum-starved cells stimulated with 20nM aPC for 15 min, washed with cold PBS and SphK1 activity assay performed according to the manufacturer instructions (Echelon, #K-3500).

Techniques: Western Blot, Fractionation, Activity Assay

Journal: bioRxiv

Article Title: aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete β-arrestin-2 mediated SphK1-S1PR1-Akt signaling axis

doi: 10.1101/2021.03.27.437291

Figure Lengend Snippet: (A) EA.hy926 cells were treated with 20 nM aPC for various times. Cells were lysed and SphK1 activity was measured using an Echelon SphK1 activity assay. The luminescence of an ATP standard curve and aPC treated cells was deteremined and the raw values are shown. Data (mean ± S.D., n = 3) was analyzed by Student’s t-test. **, P <0.001. (B) EA.hy926 cells were pretreated with or without 100 nM PF-543 and stimulated with or without 20 nM of aPC for various times. Phospho-ERK1/2 was detected by immunoblotting. Data (mean ± S.D., n = 3) was analyzed by two-way ANOVA. (B) Same as A but in HUVECs.

Article Snippet: Serum-starved cells stimulated with 20nM aPC for 15 min, washed with cold PBS and SphK1 activity assay performed according to the manufacturer instructions (Echelon, #K-3500).

Techniques: Activity Assay, Western Blot

Journal: bioRxiv

Article Title: aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete β-arrestin-2 mediated SphK1-S1PR1-Akt signaling axis

doi: 10.1101/2021.03.27.437291

Figure Lengend Snippet: ( A) EA.hy926 cells were treated with either non-specific (ns) siRNA or β-arr2 siRNA, serum-starved and then treated with 20 nM of aPC for 15 min. Cells were lysed and SphK1 activity was assessed. Data (mean ± S.D., n = 5) was analyzed by Student’s t-test. β-arr2 knockdown was verified by immunoblotting. (B) EA.hy926 cells were treated with either ns siRNA or Dvl-2 siRNA and SphK1 activity determined. Dvl-2 knockdown was confirmed by immunoblotting. Data (mean ± S.D., n = 5) was analyzed by Student’s t-test, ****, P <0.0001 and ***, P <0.001.

Article Snippet: Serum-starved cells stimulated with 20nM aPC for 15 min, washed with cold PBS and SphK1 activity assay performed according to the manufacturer instructions (Echelon, #K-3500).

Techniques: Activity Assay, Western Blot

Journal: bioRxiv

Article Title: aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete β-arrestin-2 mediated SphK1-S1PR1-Akt signaling axis

doi: 10.1101/2021.03.27.437291

Figure Lengend Snippet: Activation of PAR1 by aPC bound to its’ co-receptor EPCR promotes β-arr2-dependent SphK1 transactivation of S1PR1 that results in Akt at S473 phosphorylation, which protects endothelial cells from TNF-α induced cell death. SphK1 activation is associated with β-arr2-mediated phosphorylation, translocation to the plasma membrane following aPC stimulation.

Article Snippet: Serum-starved cells stimulated with 20nM aPC for 15 min, washed with cold PBS and SphK1 activity assay performed according to the manufacturer instructions (Echelon, #K-3500).

Techniques: Activation Assay, Translocation Assay