Horseradish peroxidase conjugated goat anti mouse

Exosome and cell pellets were lysated in lysis buffer (125 mM Tris-HCl, pH6.8, 10% sodium dodecyl sulfate (SDS), 2 M urea, 20% glycerol and 5% β-mercaptoethanol) and subjected to 10% SDS polyacrylamide gel electrophoresis and gels were transferred to a PVDF membrane. The protein was transferred to PVDF membranes, and membranes were blocked with 5% skimmed milk and probed with primary antibodies, including mouse monoclonal antibodies Alix (1:200) and CD63 (1:200), and a rabbit polyclonal antibody Cytochrome C (1:1000, Santa Cruz Biotechnology, US). The bound primary antibody was detected by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (1:5000; Sigma, USA), respectively. To examine the expression of dystrophin, protein extraction and Western blot were carried out as previously described (Han et al, 2016 (link)). Briefly, various amounts of protein from C57BL6 mice as a positive control and 50 µg total protein from muscles of treated or untreated mdx mice were used unless otherwise specified. Each experiment was performed at least three times (at least three animals).

HEK cell lines were previously described (Song et al., 2014 (link)). HeLa (Cat#ATCC-CCL-2, CVCL_0030), MDA-MB231 (Cat#ATCC-HTB-26, CVCL_0062), and MCF7 (Cat#ATCC-HTB-22, CVCL_0031) were purchased from ATCC (Manassas, VA). All cell lines were verified mycoplasma free every two months using Hoechst staining. Antibodies used were monoclonal antibodies 4C4 against ppGalNAc-T2 and UH5 against ppGalNAc-T3 (8, 31, 36), monoclonal 9e10 against the myc epitope (Evan et al., 1985 (link); Jesch et al., 2001 (link)), a polyclonal against the FLAG epitope (Bethyl Labs, Cat#A190-102B, AB_1944186), a polyclonal against GPP130 (Puri et al., 2002 (link)), a purchased anti-ppGalNAc-T3 antibody (ThermoFisher, Cat#PA5-25217, AB_2542717), an anti-α-tubulin antibody (Biolegend, Clone TU-01, Cat#625902, AB_2210041), Alexa 488 anti-mouse (Cat#A28175, AB_2536161) and Alexa 555 anti-rabbit (Cat#A27039, AB_2536100) from Thermo Fisher (Pittsburgh, PA), and horse radish peroxidase-conjugated goat anti-mouse (Cat#170–6516, AB_11125547) and goat anti-rabbit (Cat#170–6515, AB_11125142) antibodies from Sigma-Aldrich (St. Louis, MO).

Rabbit monoclonal anti α-SG (AB189254) was from Abcam (Cambridge, UK); mouse monoclonal antibodies specific for β-SG, δ-SG and γ-SG were from Leica Biosystem (Wetzlar Germany) mouse monoclonal anti β-actin was from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal antibody specific for α- and δ-SG were produced as previously described [17 (link)]. Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA).