Ecl prime western detection reagent

HASMC were harvested, and total soluble proteins were run on SDS-PAGE gels and transferred onto nitrocellulose membranes. For MMP2 experiments, the supernatant was collected and concentrated with Amicon 15-30 K columns, and then total proteins were run on a gel and transferred onto membranes. Isolation of membrane proteins was previously described^{47 (link)}. The membranes were cut and blotted for relevant proteins using specific primary antibodies (as described for each experiment). Secondary antibodies were HRP-conjugated and the chemiluminescence reaction was triggered using the Amersham ECL Prime Western detection reagent. Membranes were exposed to autoradiography films, which were scanned using a Canon scanner. Alternatively, membranes were digitalized using GE LAS 4000 mini. Quantification of the digital images obtained was performed using ImageJ (version 1.52a). For Figs. 1B, 6B and 7A,G, images were processed according to digital image and integrity policies of Nature. Increase in contrast was applied equally across the entire image for a better visualization.

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.