Ab45047

Transverse frozen sections were cut from the mid-belly of the TA or diaphragm muscle strips at a thickness of 8 μm, mounted on slides and stored at −80 °C until analysis. Immunohistochemistry for ADAMTS1 (Origene, TA317919, Rockville, MD, USA), ADAMTS5, ADAMTS15 (Abcam, ab45047, Cambridge, MA, USA), V0/V1 versican (anti-GAGβ; Merck Millipore, AB1033, Bayswater, VIC, Australia) or versikine (anti-DPEAAE neo-epitope; Thermo Fisher Scientific, PA1-1748A, Scoresby, VIC, Australia) were performed as previously described [9 (link),32 (link)]. An anti-desmin rabbit polyclonal antibody (Abcam, ab15200, Cambridge, MA, USA) together with an anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, R37116,) were used to detect myoblasts and newly regenerated myofibres [93 (link),94 (link)]. Representative wild type and mdx TA and diaphragm muscle cross-sections were H and E stained for muscle architecture, and wheat germ agglutinin to assess fibrosis [95 (link)]. For analysis of V0/V1 versican and versikine immunoreactivity, four non-overlapping representative digital images were captured with a confocal microscope of each muscle section at 600× magnification (Olympus Fluoview FV10i) and analysed for area of immunoreactivity using Image-Pro Plus software (Version 7, Media Cybernetics, Silver Spring, MD, USA).

Transfected AGS and MKN45 cells were lysed using a lysis buffer supplemented with protease inhibitors. Forty micrograms of protein were separated by 12% sodium-dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking, the membranes were incubated with the primary antibody against ADAMTSL5 (ab45047, 1: 2,500, Abcam, USA) at 4°C overnight [24 (link)], followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (ab6721, 1: 5,000, Abcam) for 2 h at room temperature. The blots were visualized using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.