Pngase f prime

Trifluoroacetic acid, Harris-modified hematoxylin, and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Sigma Aldrich (St. Louis, MO). HPLC grade methanol, ethanol, acetonitrile, xylene, hydrogen peroxide and water were obtained from Fisher Scientific (Pittsburgh, PA). Tissue Tack microscope slides were purchased from Polysciences Inc (Warrington, PA). Citraconic anyhydride and SafeClear II was purchased from Thermo Scientific (Bellefonte, PA). Recombinant Peptide N-Glycosidase F (PNGase F) from Flavobacterium menigosepticum was obtained, expressed, and purified as previously described [34 (link)], but is also available commercially as PNGase F Prime™ from Bulldog Bio (Portsmouth, NH). Universal Antigen Retrieval Reagent was purchased from R&D Systems (Minneapolis, MN).

The levels of proteins constitutively expressed in EVs from different types parent cells were identified by Western blot analysis, using Wes^™ Simple Western Blot device (ProteinSimple, San Jose, CA, USA). EVs were lysed with 1x RIPA buffer for 30 minutes at room temperature and 200 or 40 mg/mL of protein was denatured and loaded in Wes^™ multi-well plates following manufacturer’s instructions. The expression levels of EV-specific proteins were also examined in parent cells in control experiment. Protein concentrations were determined using BCA kit (Pierce Biotechnology, Rockford, IL, USA). For analysis of CD9, sample’s lysates de-glycosylated using PNGase F PRIME (Bulldog Bio, NZPP050) under non-denaturing conditions at the ration 1:9 v:v for 1 h prior to denaturalization. The protein bands were detected with primary antibodies described in Supplementary Table S3, and secondary Goat Anti-Rabbit HRP Conjugate (ready-to-use reagent, ProteinSimple, San Jose, CA, USA). The protein concentration for all samples were kept the same, 200 μg/well. A quantitative analysis of obtained images was carried out using Compass SW software.