Stx12 is required for normal α-granule biogenesis. (A) IB analysis of total extracts from imMKCL cells treated with either Control or Stx12 siRNA. (B) Quantification of the experiment is shown in A (n = 3). Stx12 siRNA samples were compared with Control siRNA, and statistical significance was determined via unpaired, 2-tailed Student t test. (C) Immunofluorescence microscopy analysis of wild-type (WT) or Stx12 KO imMKCL cells costained with antibodies against P-selectin, PF4, and TGN38. Bar = 5 µm. (D) Fluorescence intensity quantification of the experiment shown in C (WT, n = 50; Stx12 KO, n = 59). Stx12 KO samples were compared with WT samples, and statistical significance was determined via unpaired, Mann-Whitney U test. A.U., arbitrary units. (E) Thin-section transmission electron microscopy analysis of WT and Stx12 KO imMKCL cells subjected to high-pressure freezing. Original magnification, ×11 000 for the picture showing the entire cell and ×49 000 for the enlarged insets (bars represent 1 μm and 100 nm, respectively). (F) Quantification of α-granules, δ-granules, and MVBs visualized in the experiment shown in E (n = 12 cells per genotype). Stx12 KO samples were compared with WT samples, and statistical significance was determined via unpaired, Mann-Whitney U test. ν, nucleus; α, α-granules; δ, δ-granules; MVB, multivesicular body; mi, mitochondria.