![Figure 5. Abcc4 KO platelets have decreased GPVI expression. (A) Immunoblots of whole-cell lysates prepared from Abcc4 WT and KO platelets, loaded at the indicated concentrations, and probed with either GPVI or its binding partner FcRγ. (Β) After densitometry and normalization for loading, the signals were quantified (*P < .05). (C) Immunoblot analysis of total-platelet lysate and surface membrane proteins determined by biotinylation of mouse Abcc4 WT and KO platelets. The transferrin receptor (TFR) was used as a membrane control. (D) After densitometry and normalization by total lysate (Total), the percent on the membrane surface was determined. The error bar represents the standard error of the mean of 3 experiments. (E) Immunoblots of whole-cell lysates prepared from Abcc4 WT and KO platelets loaded at the indicated concentrations and probed with Plcγ2, Syk, and actin antibodies. (F) After normalization by actin, the relative amount of Plcγ2 and Syk is shown. (G) Basal cAMP levels in Abcc4 WT and KO platelets measured by competitive enzyme-linked immunosorbent assay (*P < .05. (H) Ratio of internal vs external cAMP levels post–PGE1 stimulation in the Abcc4 WT and KO platelets measured by competitive enzyme-linked immunosorbent assay (*P < .05). (I) Immunoblot of platelet-surface membrane proteins obtained after surface biotinylation and avidin-bead pull-down from Abcc4 WT platelets, either untreated or treated with 10 μM of forskolin. Immunoblots were probed with GPVI, as the loading control blot was stained with ponceau S (I, input from total lysate; B, bound to the avidin-bead [ie, surface protein and S in supernatant or cytosol]). (J) Representative Vasp and phospho-Vasp immunoblots in platelet lysates prepared from Abcc4 WT and KO treated with either 1 or 2 μg of collagen, 0.01 U of thrombin, or 10 μM of forskolin. Blots were probed with p-Vasp Ser 157, p-Vasp Ser 239, and an antibody that recognizes total Vasp. Phosphorylation was determined for Ser 157 (K) or Ser 239 (L) as percentage of forskolin (Fsk)-induced phosphorylation. Platelet lysate immunoblot analysis is performed 3 times using pooled PRP-derived platelets from 2 to 4 mice per genotype. The bar in the graph represents 1 standard deviation. Untxt, untreated.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/126/20/10.1182_blood-2014-08-595942/4/2307f5.jpeg?Expires=1724254035&Signature=ItcMYwS45kd2RPBS3nt3K4BGdMMjDv9G8SCV98hXPmHXVajI99XVRTVui1hrzoHdcfTDoejyQb6yusf~IJDMmfQ1u36HW3Ju~R2RqL1f-zRsA4zRZV2oLEj~VW3FWzx8Z3yZW~wQMnUdJuQNGmoYYHofgE63B2oPgjQljJdYMm5~pVGjA~peqkTtLa8zivokxuWT6XmOUAKgJkCbjXAHEPiPDsirArlTL~8ZSlNBVE-xFYaokQtPNPZZtqakKTekVyOYkhxJX08IUFBGOHWnLRIOjtt~lwtM8m7Wa2UFQfgbC6JoYlhEyDNv1E7YihS4EAijF25lsGXTV-15hz5HVQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Abcc4 KO platelets have decreased GPVI expression. (A) Immunoblots of whole-cell lysates prepared from Abcc4 WT and KO platelets, loaded at the indicated concentrations, and probed with either GPVI or its binding partner FcRγ. (Β) After densitometry and normalization for loading, the signals were quantified (*P < .05). (C) Immunoblot analysis of total-platelet lysate and surface membrane proteins determined by biotinylation of mouse Abcc4 WT and KO platelets. The transferrin receptor (TFR) was used as a membrane control. (D) After densitometry and normalization by total lysate (Total), the percent on the membrane surface was determined. The error bar represents the standard error of the mean of 3 experiments. (E) Immunoblots of whole-cell lysates prepared from Abcc4 WT and KO platelets loaded at the indicated concentrations and probed with Plcγ2, Syk, and actin antibodies. (F) After normalization by actin, the relative amount of Plcγ2 and Syk is shown. (G) Basal cAMP levels in Abcc4 WT and KO platelets measured by competitive enzyme-linked immunosorbent assay (*P < .05. (H) Ratio of internal vs external cAMP levels post–PGE1 stimulation in the Abcc4 WT and KO platelets measured by competitive enzyme-linked immunosorbent assay (*P < .05). (I) Immunoblot of platelet-surface membrane proteins obtained after surface biotinylation and avidin-bead pull-down from Abcc4 WT platelets, either untreated or treated with 10 μM of forskolin. Immunoblots were probed with GPVI, as the loading control blot was stained with ponceau S (I, input from total lysate; B, bound to the avidin-bead [ie, surface protein and S in supernatant or cytosol]). (J) Representative Vasp and phospho-Vasp immunoblots in platelet lysates prepared from Abcc4 WT and KO treated with either 1 or 2 μg of collagen, 0.01 U of thrombin, or 10 μM of forskolin. Blots were probed with p-Vasp Ser 157, p-Vasp Ser 239, and an antibody that recognizes total Vasp. Phosphorylation was determined for Ser 157 (K) or Ser 239 (L) as percentage of forskolin (Fsk)-induced phosphorylation. Platelet lysate immunoblot analysis is performed 3 times using pooled PRP-derived platelets from 2 to 4 mice per genotype. The bar in the graph represents 1 standard deviation. Untxt, untreated.
