Western Blot Analysis of Exosomal Proteins

Cell exosomes were lysed in a western blotting lysis buffer-radioimmunoprecipitation (RIPA) with PMSF, then, the protein concentrations were quantified with a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China) [29 (link)]. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently electro-transferred onto a PVDF membranes, and the membranes were incubated in following antibodies: CD9 (rabbit IgG, proteintech), TSG101 (rabbit IgG, Abcam), HSP70 (rabbit IgG, SBI), Calnexin (rabbit IgG, CST), Alix (rabbit IgG, proteintech), GAPDH (rabbit IgG, proteintech), GAPDH was used as internal control. followed by chemiluminescent detection (Tanon, China) [30 (link)].

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Yu H., Yang X., Xiao X., Xu M., Yang Y., Xue C., Li X., Wang S, & Zhao R.C. (2021). Human Adipose Mesenchymal Stem Cell-derived Exosomes Protect Mice from DSS-Induced Inflammatory Bowel Disease by Promoting Intestinal-stem-cell and Epithelial Regeneration. Aging and Disease, 12(6), 1423-1437.

Publication 2021

BCA) protein assay kit TSG101 HSP70 GAPDH

Antibodies Assay Bicinchoninic acid Buffer Calnexin Cell Exosomes Gapdh Hsp70 Membranes Protein a Proteins Pvdf Rabbit Radioimmunoprecipitation Sds page Tsg101

Corresponding Organization : Chinese Academy of Medical Sciences & Peking Union Medical College

Other organizations : Shanghai University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein concentrations
Protein expression levels of CD9, TSG101, HSP70, Calnexin, Alix, and GAPDH

control variables

Cell exosomes were lysed in a western blotting lysis buffer-radioimmunoprecipitation (RIPA) with PMSF
Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Proteins were electro-transferred onto PVDF membranes
GAPDH was used as an internal control

controls

Positive control: None mentioned
Negative control: None mentioned

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