Horseradish peroxidase conjugated anti rabbit igg and anti mouse igg

Total cell lysates were resolved on 4%–12% bolt protein gels by SDS-PAGE (Bolt Bis-Tris Plus gels, Thermo Fisher Scientific) and transferred to nitrocellulose membrane (Millipore). The membrane was blocked for 1 h in 3% nonfat dry milk in Tris-buffered saline (TBS) with 0.2% Tween 20 and incubated in primary antibodies overnight at 4°C. Incubation was performed with the following primary antibodies: dystrophin C-terminal (NCL-Dys1, 1:100, Leica Biosystems) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MAB374, 1:50,000, Chemicon). Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG (GE Healthcare) secondary antibodies were used at 1:15,000 dilutions in 3% nonfat dry milk and incubated at room temperature (RT) for 2 h. Immunoblots were developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific).

Equal quantities of protein samples were resolved on 4-20% Precise Protein Gels by SDS-PAGE (4-20% Precise Protein Gels, Thermo Fisher Scientific) and transferred to nitrocellulose membrane (Millipore). Membranes were blocked for 1 hour in 5% nonfat dry milk in TBS with 0.2% Tween 20 and incubate in primary antibodies overnight at 4 °C. Incubations were performed with the following primary antibodies: dystrophin (MANDYS1; 1:5), utrophin (MANCHO3; 1:50), α-DG IIH6 (sc-53987; 1:500; Santa Cruz Biotechnology, Inc.), β-DG (MANDAG2; 1:250), α-SG (VP-A105; 1:100), β-SG (VP-B206; 1:100), γ-SG (VP-G803; 1:100), nNOS (A-11, 1:250; Santa Cruz), laminin (L9393; 1:5,000), β1D integrin (MAB1900; 1:100), GAPDH (MAB374, 1:50,000; Chemicon) and SSPN (E2; 1:500; Santa Cruz). Horseradish peroxidase–conjugated anti–rabbit IgG and anti–mouse IgG (GE Healthcare) secondary antibodies were used at 1:2,000 dilutions in 5% nonfat dry milk and incubated at RT for 3 h. Immunoblots were developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific). Mean integrated density value was quantified using ImageJ (NIH).