Western Blot Analysis of Drosophila Mkrn1

We used our previously generated antibody against Drosophila Mkrn1 [37 (link)]. Protein extracts from the head, thorax, abdomen, and ovaries of adult female Drosophila were prepared using lysis buffer (10 mM HEPES [pH 7.5]; 50 mM KCl; 10% glycerol; 5 mM Tris-HCl [pH 7.5]) with freshly added 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, protease inhibitor (Sigma), 1 mM Na₃VO₄, and 0.25 mM NaF (final concentration). For p-AKT and p-S6K analysis, phosphatase inhibitor cocktail 2 and 3 (Sigma) were used instead of Na₃VO₄ and NaF. The protein extracts were resolved by SDS-PAGE and the resulting blots were probed using the following primary antibodies: rabbit anti-Mkrn1, 1:3000 [37 (link)]; rabbit anti-phospho-AKT (Ser505), 1:1000 (Cell Signaling Technology, 4054); rabbit anti-AKT, 1:1000 (Cell signaling Technology, 9272); rabbit anti-phospho-S6K (Thr398), 1:1000 (Cell Signaling Technology, 9209); guinea pig anti-dS6K, 1:3000 [38 (link)]; mouse anti-Armadillo, 1:1000 (DSHB, N2 7A1); rabbit anti-p44/42 MAPK (Erk1/2), 1:2000 (Cell Signaling Technology, 9102). Band intensities were quantified using ImageJ software.