Bs 6934r

Ten µg protein from the indicated SEC fractions (concentrated by centrifugal filtration), pellets, or MFGM preparation were separated on NuPAGE Novex Gels, 10% Bis-Tris (MOPS buffer) under non-reduced conditions. The separated proteins were transferred to PVDF membranes (1 h at 200 V and 500 mA). PVDF membranes were blocked in 2% Tween-20 in TBS (50 mM Tris, 0.5 M NaCl) pH 7.4 and probed with the following primary antibodies: in-house produced rabbit anti-bovine PAS-7/lactadherin (polyclonal), and rabbit anti-human BA46/lactadherin (polyclonal) produced by MedProbe.com targeting the human lactadherin peptides CEE ISQEVRGDVFPSY and DSANWTEYQDPRTGS. Both human and bovine lactadherin appears in two glycosylation variants,[37 ] and, therefore, two bands of the protein are detected. Bovine lactadherin (PAS-6/7) [37 ] and human lactadherin (BA46) [38 (link)] standards were purified as previously described, and stored in 75 mM sodium phosphate, pH 7.0 at −80°C. Purchased antibodies include mouse anti-human CD63 (TS63, Abcam, Cambridge, UK), mouse anti-human CD9 (H19a, BioLegend, San Diego, CA, USA), rabbit anti-human CD81 (bs-6934R, Bioss Antibodies, Woburn, MA, USA), rabbit anti-human Beta-casein (Ab112595, Abcam).

The protein samples were extracted from tissues, cells and exosomes using radio-immunoprecipitation assay buffer (Beyotime). Equal amount (25 μg) of protein was separated through SDS-PAGE and blotted onto polyvinylidene fluoride membranes (BIO-RAD). Then, blocking in 5% skim milk was carried out. Proteins were detected by incubation with appropriate primary antibodies TFIIB (1:500, A1708, ABclonal, Wuhan, China), LaminA/C (1:10000, A19524, ABclonal), CD63 (1:500, bs-0342R, Bioss), CD81 (1:500, bs-6934R, Bioss), Hsp70 (1:500, bs-0244R, Bioss), TSG101 (1:500, A5789, ABclonal), YKL-40 (1:500, bs-10215R, Bioss), STAT3 (1:1000, bs-52235R, Bioss), p-STAT3 (1:1000, bs-22386R, Bioss), Rab27a (1:500, A1934, ABclonal), β-actin (1:20000, AC026, ABclonal) overnight at 4°C coupled with HRP-conjugated Goat Anti-Rabbit secondary antibody (1:5000, AS063, ABclonal). ECL reagent (Solarbio, Beijing, China) was used for visualization of proteins.