Rabbit anti flag tag

Protein was extracted from differentiated myoblasts using NCH buffer (4% sodium dodecyl sulphate, 4M urea, 150 mM Tris) diluted 1:1 with RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma) containing a complete protease inhibitor cocktail (1:100, Roche). Samples were collected and boiled for 3 minutes and 30 μl of each sample was run on a NuPAGE Novex 3–8% Tris-Acetate Gel, with constant voltage of 150 V for 1 hour, before transfer to a nitrocellulose membrane at 300 mA for 2 hours. The membrane was blocked in Odyssey blocking solution (LI-COR Biosciences) for 60 min, before incubation with rabbit anti-dystrophin (1:2000, Fisher Scientific) or rabbit anti-FLAG-tag (1:2000, Sigma) and mouse anti-beta-actin (1:4000, Invitrogen) overnight at 4 °C. The membrane was washed with PBS containing 1% Tween 20 (PBST) before incubation with IRDye 680 RD goat anti-rabbit and IRDye 800CW goat anti-mouse 2nd antibodies (1:15000, LI-COR Biosciences) for 1 hour at room temperature. The fluorescent image was acquired by Odyssey Clx infrared imaging system (LI-COR Biosciences) using image studio software 3.1.4.

For immunofluorescence staining, number 4 mammary glands were fixed in 10% formalin for 24 h, moved to 70% ethanol solution for 24 h, and embedded in paraffin. Then, 5 μm sections were cut and dehydrated in xylene and an ethanol gradient. After performing antigen retrieval in 10 mM citrate sodium containing 0.05% tween-20, the sections were washed with PBS and incubated with primary antibody overnight at 4 °C in antibody solution containing 10% donkey serum and 0.05% tween-20. Slides were washed three times in PBS containing 0.05% Triton X-100 and incubated with secondary antibody in antibody solution for 1 h at room temperature in the dark. Slides were washed in PBS, incubated with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature, washed in PBS, mounted with coverslips using ProLong^® Gold antifade reagent (Life Technologies, Carlsbad, CA, USA). Slides were imaged on a Leica DMI 6000B fluorescence microscope. The primary antibodies used are Rat anti-Cytokeratin 8 (DSHB) and Rabbit anti-Flag-tag (Sigma). The secondary antibodies were conjugated with either Alexa fluor 488 or Alexa fluor 568 (Molecular probe).