Western Blot Analysis of Autophagy Markers

Western blot analysis was conducted as previously described44 (link). The following primary (at a concentration of 1.5 μg ml⁻¹) and secondary (0.5 ng ml⁻¹) antibodies were used for western blotting: polyclonal anti-ARRDC1, anti-LC3, anti-MDA5, anti-TLR9 (Abcam), polyclonal anti-Atg-12 (Santa Cruz Biotechnology, SC-68884), polyclonal anti-Pink1 (Santa Cruz Biotechnology, SC-33796), anti-Parkin mouse monoclonal (clone PRK8, Santa Cruz Biotechnology), polyclonal anti-Miro (Santa Cruz Biotechnology, SC-292547), anti-CD9 (MM2/57, Millipore), anti-CD63 (MEM-259, ThermoFisher), anti-TSG101 (51/TSG101, BD Biosciences), anti-Human MFGE8 (Abnova), anti-Dicer (D11, Santa Cruz Biotechnology, used at a concentration of 2 μg ml⁻¹), anti-Tubulin, anti-LC3 (rabbit polyclonal), anti-TLR-7 (Cell Signaling Technology), Beta actin (AC-15, Sigma-Aldrich) and anti-GAPDH (Santa Cruz Biotechnology, SC-25778). NF-κB nuclear translocation was characterized by staining cells with anti NF-κB-P50 Alexa Fluor 488 (E-10, Santa Cruz Biotechnology). Images were obtained using a Leica TCS NT upright confocal microscope.

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Phinney D.G., Di Giuseppe M., Njah J., Sala E., Shiva S., St Croix C.M., Stolz D.B., Watkins S.C., Di Y.P., Leikauf G.D., Kolls J., Riches D.W., Deiuliis G., Kaminski N., Boregowda S.V., McKenna D.H, & Ortiz L.A. (2015). Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nature Communications, 6, 8472.

Publication 2015

anti-ARRDC1 anti-LC3 anti-MDA5 anti-TLR9 anti-Atg-12 anti-Pink1 anti-Parkin mouse monoclonal (clone PRK8, anti-Miro anti-CD9 (MM2/57, anti-CD63 (MEM-259, anti-Human MFGE8 anti-Dicer (D11, anti-TLR-7 Beta actin (AC-15, anti-GAPDH anti NF-κB-P50 Alexa Fluor 488 (E-10, TCS NT upright confocal microscope

Alexa fluor 488 Antibodies Beta actin Cells Clone Confocal microscope Dicer Gapdh Human Mda5 Mfge8 Mouse Nf κb Nf κb p50 Parkin Rabbit Translocation Tsg101 Tubulin Western blot analysis

Corresponding Organization :

Other organizations : Scripps Research Institute, University of Pittsburgh, Hospital Universitario Son Espases, Richard King Mellon Foundation, National Jewish Health, University of Colorado Denver, Yale University, University of Minnesota

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Variable analysis

independent variables

Concentration of primary antibodies (1.5 μg ml^-1)
Concentration of secondary antibodies (0.5 ng ml^-1)

dependent variables

Expression levels of ARRDC1, LC3, MDA5, TLR9, Atg-12, Pink1, Parkin, Miro, CD9, CD63, TSG101, MFGE8, Dicer, Tubulin, TLR-7, Beta actin, GAPDH
NF-κB nuclear translocation

control variables

Western blot analysis protocol (as previously described in reference 44)
Microscope used for imaging (Leica TCS NT upright confocal microscope)

positive controls

None specified

negative controls

None specified

Annotations

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