SLPI is expressed in megakaryocytes, binds specifically to β1 tubulin, and is reduced in platelets lacking β1 tubulin. (A) SLPI binds to β1 tubulin but not to the homologous β5 tubulin isotype in yeast 2-hybrid assays. Cells transformed with β5 tubulin fail to grow on His^-Ade^- selection medium, whereas growth of β1 tubulin-transformed cells indicates that SLPI and β1 tubulin interact and activate the reporter genes. Other transformants (not shown) with β5 tubulin grew on His^-Ade^- media and served as positive controls. (B) Immunofluorescence (IF) analysis of primary MKs expressing a β1 tubulin-GFP fusion protein. Cellular SLPI is detected by a specific antibody and Texas red-labeled secondary antibody. Merger of IF signals, including nuclear DAPI staining, is displayed in the third column, and each row represents different cells. Scale bars, 10 μm. (C) Double-label IF of primary MKs with β-tubulin (green) and SLPI (red) Ab, showing significant colocalization in the merged image. (D) SLPI mRNA is expressed in normal (p45^+/?) primary MKs and reduced in p45 NF-E2^-/- cells, as judged by semiquantitative RT-PCR over the indicated number of PCR cycles. (E) SLPI appears as a 14-kDa protein in platelet lysates, as shown by immunoblotting using a specific antibody. No signal is detected in SLPI^-/- platelet lysates. (F-G) SLPI expression is reduced in β1 tubulin^-/- platelets, whereas absence of SLPI does not affect β1-tubulin levels. Equal loading of wild-type and mutant platelets was verified by staining with antisera against whole mouse platelets (RAMPS) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).