Tsg101 sc 7964

The protein-based concentration of EVs, CM and Sup was measured using Pierce BCA Protein Kit (Cat # 23225, ThermoFisher Scientific) for bicinchoninic acid assay according to the protocols provided by the manufacturer. Samples (40 µg proteins) were separated on 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) by applying 120 Volt (V) in running buffer for 90 min. In the next step, proteins were transferred to the nitrocellulose membranes by applying 110 V in transfer buffer for 70 min. Membrane blocking was done with 5% skimmed milk prepared in Tris-buffered saline containing 0.1% Tween 20 (1 × TBST). Incubation of membranes with primary antibodies was performed overnight at 4 °C. Primary antibodies included: TSG101 (SC-7964, Santa Cruz Biotechnology) with 1:200 dilution, CD81 Antibody (SC-166029, Santa Cruz Biotechnology) with 1:200 dilution and CD9 (Ab92726, Abcam) with 1:1000 dilution. Afterwards, membranes were incubated for 1 h with respective secondary antibodies (HRP conjugated antibodies) (Dako, Agilent Denmark).

Western blot analysis was carried out as previously described [12 (link)]. Due to the limited lane number available on PAGE gel, the analyzed LCC were reduced to two lines. Antibodies to AchE (SC-11409, 1:1000), TSG101 (sc-7964, 1:1000), CD81 (sc-166029, 1:1000), and Hsp70 (SC-24, 1:1000) were from Santa Cruz Biotechnology. Antibody to Hsp90 (13171-1-AP, 2:1000) was from Proteintech. Antibody to CD63 (A5271, 1:1000) was from ABclonal (Woburn, MA, USA). Antibodies to total (9212L, 1:2000) and phosphorylated p38 MAPK (4511S, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies for MHC (MF20, 1:5000) were from Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA. Antibody for Atrogin1/MAFbx (1:2000) was custom-generated by Pocono Rabbit Farm & Laboratory and verified previously (Zhang et al., 2021). Antibodies for UBR2 (NBP1-45243, 1:1000) and LC3 (NB100-2220, 1:1000) were from Novus Biologicals (Littleton, CO, USA). Antibodies for total C/EBPβ (3082S, 1:1000) and C/EBPβ phosphorylated on Thr-235 (3084, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA). Antibody for β-actin (sc-47778, 1:5000) was from Santa Cruz Biotechnology (Dallas, TX, USA). The optical densities of the detected bands were normalized to loading control β-actin or Ponceau S-stained proteins, except for LC3-II, which was normalized to LC3-I.

Radio-immunoprecipitation assay buffer (SenBeiJia Biological Technology Co., Ltd., Nanjing, China) was applied to collect total protein from tissues and cells. The protein concentration was measured bicinchoninic acid protein assay kit (Beyotime, China). About 40 μg of protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, electro-blotted onto PVDF membrane (Millipore, USA) and blocked with 5% skimmed milk. Antibodies were showed as follows: anti-CXCL17 (AF4207, 1:1000) from R&D Systems (MN, USA), CD63 (sc-5275, 1:1000) and TSG101 (sc-7964, 1:1000) from Santa Cruz Biotechnology (CA, USA), Alix (2171, 1:1000) from Cell Signaling Technology (MA, USA), and horseradish peroxidase-conjugated secondary antibody (1:10,000). The protein expression was detected by Quantity-one software (Bio-Rad Laboratories, USA) with enhanced chemiluminescence kit [26 (link)].

Antibodies against p-JNK (#9255), JNK (#9252), cleaved caspae-3 (#9661), cleaved caspae-9 (#9509), p-Ser-Pro (#2325), and p-Thr-Pro (#9391) were from Cell Signaling Technology (Beverly, MA) and used at a dilution of 1:1,000 for immunoblot analysis. Antibodies against Bax (sc-20067, diluted to 1:1,000), CD63 (sc-15363, diluted to 1:1,000), TSG 101 (sc-7964, diluted to 1:1,000), and β-actin (sc-47778, diluted to 1:2,000), were from Santa Cruz Biotechnology (Dallas, TX). Antibodies against CYP2E1 (ab28146, diluted to 1:3,000), 3-NT (sc-ab6139, diluted to 1:3,000), and iNOS (ab136918, diluted to 1:1,000), were from Abcam (Cambridge, Mass).