Figure 2. P2Y₁₁ protein and distribution in arterial myocytes.

(A) Representative blot of immunoreactive bands of expected molecular weight for endogenous P2Y₁₁ (~40 kDa), overexpressed P2Y₁₁-GFP (~70 kDa), and β-actin (~43 kDa) in untransfected, vehicle-treated (empty transfection) and P2Y₁₁-GFP transfected tsA-201 cells (n = 3 lysates per condition). Note that tsA-201 cells endogenously express P2Y₁₁ (Dreisig and Kornum, 2016). (B) Representative blot of immunoreactive bands of expected molecular weight for endogenous P2Y₁₁ (~40 kDa), overexpressed P2Y₁₁-GFP (~70 kDa), and β-actin (~43 kDa) in tsA-cells transfected with P2Y₁₁-GFP as well as corresponding P2Y₁₁ sense (SNS) or antisense (ANS) ODNs (64% reduction in endogenous P2Y₁₁ expression in cells treated with ANS; 62% reduction in P2Y₁₁-GFP expression in P2Y₁₁-GFP-transfected cells treated with ANS; n = 3 lysates per condition; Figure 2—source data 1). (C) Representative immunoblot detection of P2Y₁₁ (~40 kDa) in lysates from human and wt mouse arteries (n = 3 arterial lysates per sample). (D) Representative confocal images of P2Y₁₁-associated fluorescence (green), wheat germ agglutinin (WGA, red) and merged channels in human (n = 11 cells from three humans) and wt mouse (n = 14 cells from three mice) arterial myocytes. (E) Line profile of the P2Y₁₁- and WGA-associated fluorescence from the area highlighted by the dotted lines in the representative human and mouse arterial myocytes in D.

Figure 2—figure supplement 1. Full-length blot for Figure 2C, knock down of P2Y₁₁ in arterial lysates, P2Y₁₁ immunoreactivity in isolated mouse arterial lysates, negative controls for immunofluorescence experiments in Figure 2D and P2Y₁₁ antibody control.

(A) Full-length blot corresponding to Figure 2C. Red box indicates the crop region displayed in main figure. (B) Representative blots of immunoreactive bands of expected molecular weight for P2Y₁₁ (~40 kDa) in human (n = 5 arterial lysates per condition) and mouse (n = 5 arterial lysates per condition) arterial lysates treated with P2Y₁₁ sense (SNS) and antisense (ANS) ODNs (*p<0.05, Wilcoxon matched pairs test; Figure 2—figure supplement 1—source data 1). (C) Representative confocal images of human (left; n = 10 cells from two humans) and mouse (right; n = 10 cells from two mice) arterial myocytes in which the primary antibody for P2Y₁₁ (no 1° Ab) was excluded from the preparation (e.g. negative control). Wheat germ agglutinin (WGA, red) was used to label the plasma membrane. (D) Representative blots of immunoreactive bands of expected molecular weight for P2Y₁₁ (~40 kDa) and total protein in isolated mouse arterial myocyte lysates (n = 2 lysates). (E) Exemplary confocal images of P2Y₁₁-associated fluorescence (left) and wheat germ agglutinin (WGA, right) in wt mouse arterial myocytes stained with unboiled (n = 7 cells from six mice) or boiled (n = 7 cells from six mice) P2Y₁₁ primary antibody.