Standard western blot protocol

Muscle tissue was homogenized using a Bullet Blender (Next Advance, NY USA) in CellLytic MT buffer (Sigma Aldrich) using 0.5 mm Zirconium oxide beads. The supernatant was aliquoted and utilized for Bradford assay (Beckman Coulter DU 730 spectrophotometer at 595 nm) to determine total protein content. Standard Western Blot protocol (Abcam) was performed using 8 ul of sample and ladder (Bio-Rad) loaded into SDS gel wells (ClearPAGE; 4–20%), run in a Tris-Tricine SDS running buffer (ClearPage) and transferred using Tris/Glycine buffer (Bio-Rad). Three primary antibodies specific to the myosin heavy chains I, IIa and IIb (Developmental Studies Hybridoma Bank, University of Iowa, BA-D5 (1:750), SC-71 (1:500) and BF-F3 (1:1) respectively) and two secondary antibodies [KPL peroxidase labeled goat anti-mouse IgG (H+L) at 1:10,000, Invitrogen HRP goat anti-mouse IgG+A+M at 1:30,000] were used. Primary antibodies have been previously confirmed for fiber typing in pinnipeds (Watson et al., 2003 (link); Kanatous et al., 2008 (link)). Protein bands on a nitrocellulous membrane were visualized using a chemiluminescent substrate kit (KPL International) sensitive to peroxidase-labeled antibodies and developed using a luminescent image analyzer (GE LAS 4000) and accompanying software (GE Healthcare Life Sciences). Western Blot analysis was completed in duplicate.

We used a strong RIPA lysis buffer (CW-BIO, Jiangsu, China) and the PMSF (Phenylmethylsulfonyl fluoride) protease inhibitor (Beyotime, Shanghai, China) to obtain protein samples from the head tissues of mutant flies. Western blot was performed according to standard western blot protocol (Abcam, Cambridge, UK). We used primary antibodies to Dscam1 (ab43847, diluted 1:5,000), β-actin (ab8227, diluted 1:10,000), and secondary antibodies (goat anti-rabbit IgG, 1:10,000, CW-BIO) in this process. The immunoreactive bands of Dscam1 and β-actin protein were detected using the eECL Western Blot Kit (CW-BIO) and Tanon 5200. Semiquantitative western blot analysis compared the Dscam1 protein levels from the head tissues of different genotypes. Image J software (NIH, Maryland, USA) was used to analyze quantitatively the western blot bands.