Immunoblotting Analysis of Larval Muscle Proteins

Third-instar larval muscle extracts (12 dissected body walls of each genotype) were prepared and used for immunoblotting, as previously described^{36 (link)}. Larval body wall muscles were homogenized in ice-cold RIPA buffer (Cell Signaling Technology) mixed with an EDTA-free protease inhibitor cocktail (Thermo Scientific) and run on 4-12% Bis–Tris Plus gels. After blotting onto PVDF membrane (Novex) and incubated with 5% nonfat milk in TBST (Thermo Scientific, with 5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary antibodies at 4°C overnight. The following antibodies were used: mouse anti-DLG (4F9, 1:1000, Developmental Studies Hybridoma Bank, USA), guinea pig anti-CaMKII (1:1000, this study), mouse anti-β-tubulin (E7, 1:200; Developmental Studies Hybridoma Bank, USA). Membranes were washed three times and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-guinea pig secondary antibodies (Jackson ImmunoResearch) for 1 h. Blots were washed with TBST and visualized with the ECL Prime Western Detection Reagent (Amersham) and exposed to G:BOX Chemi XX6 (Syngene). Bands intensities were determined with ImageJ (NIH) using the gel analysis plug-in.