Western Blot Analysis of Sima and Msi Proteins

Protein extracts from S2R+ cells or embryos (stage E14 to stage E17) were prepared in RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) SDS, 0.5% (w/v) sodium deoxycholate and 1% Triton X-100) with the addition of proteinase inhibitory cocktail (Invitrogen) and kept at 4°C. 25–50 μg of total extracts were loaded on a 6–11% polyacrylamide gel, subjected to electrophoresis and then blotted onto nitrocellulose membrane (Bio-Rad). Thereafter, membranes were blocked for 1 h at room temperature with 5% nonfat milk or BSA in TBS with 0.1% Tween 20 (TBS-T) and incubated overnight with rabbit anti-Sima (29 (link)); rat anti-dMsi 3A5 (26 (link)) or mouse anti-tubulin (Invitrogen) in 5% nonfat milk in TBS-T. The secondary antibodies used were HRP conjugated (1/5000, Jackson ImmunoResearch). Immunoblots were developed with the ECL prime detection reagent (Amersham).