Fig. 5: Deletion of hyperactive β1 integrin restores thrombopoiesis by B4galt1^−/− MKs.

a Total BM-derived MK and blood platelet lysates obtained from control and B4galt1^−/− mice were subjected to SDS–PAGE. Immunoblots were probed with mAbs directed against β1 and β3 integrin subunits, GPIbα, αIIb, α5, α6 integrin subunits, and GAPDH as loading control. b Densitometric quantification of immunoblots probed with antibodies directed against the β1 and β3 integrin subunits and GPIbα of control (white) and B4galt1^−/− (green) MKs and platelets. n = 3 in each group. Data are expressed as mean ± SEM. Groups were compared using an unpaired Student’s t-test, *p < 0.05. c Control and B4galt1^−/− platelet lysates, treated (+) or not (−) with PNGaseF (PNGF) to remove all N-linked glycans were subjected to SDS–PAGE and immunoblots were probed with an anti-β1 integrin antibody. d Total platelet lysates (input) and platelet lysates immunoprecipiated with anti-β1 integrin antibody (IP) obtained from control and B4galt1^−/− mice were subjected to SDS–PAGE. Immunoblots were probed with mAbs directed against β1 integrin subunit, ECL, and GAPDH. e Immunofluorescence of control and B4galt1^−/− mouse BM sections stained with the antibody 9EG7 directed against activated β1 integrin subunit (red) and DAPI (blue). f Quantification of red fluorescence intensity in control (white) and B4galt1^−/− MKs (green). n = 3 in each group. Data are expressed as mean ± SEM. Groups were compared using an unpaired Student’s t-test, ****p < 0.0001. g Total lysates from control, B4galt1^−/−, Itgb1^PF4+, and B4galt1^−/−Itgb1^PF4+ platelets were subjected to SDS–PAGE and probed with anti-p-Tyr, pY397 FAK, pY576 FAK, total FAK, and β-actin mAbs. Densitometric quantification of immunoblots probed with h pY397 FAK (n = 3) and i pY576 FAK (n = 6) mAb, normalized for total FAK. Data are expressed as mean ± SEM. One-way ANOVA was used to compare each group, *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.