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.
Cd81 sc 166029
Manufactured by Santa Cruz Biotechnology
Sourced in United States
CD81 (sc-166029) is a laboratory product offered by Santa Cruz Biotechnology. It is a reagent used for the detection and study of the CD81 protein, which is a member of the tetraspanin family. The core function of CD81 is to facilitate cell-cell interactions and signaling processes. This product is intended for research use only and its detailed description is limited to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Image system, by Bio-Rad (2 mentions)
Chemiluminescence kit, by Merck Group (2 mentions)
Zb 2301, by ZSGB-BIO (2 mentions)
Alix sc 53540, by Santa Cruz Biotechnology (2 mentions)
Mcu 26312 1 ap, by Proteintech (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lysis buffer, by Keygen Biotech (2 mentions)
Protease and phosphatase inhibitor cocktail solution, by Keygen Biotech (2 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Tsg101 sc 7964, by Santa Cruz Biotechnology (1 mentions)
Hsp70 sc 24, by Santa Cruz Biotechnology (1 mentions)
Antibody for β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Nbp1 45243, by Novus Biologicals (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Transmission electron microscopy, by Hitachi (1 mentions)
5 protocols using cd81 sc 166029
1
Western Blot Analysis of Muscle Proteins
2
Extracellular Vesicle Protein Analysis
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EVs, dEVs, and serum samples were solubilized with 2% SDS or RIPA buffer and thoroughly vortexed. Protein concentration was determined using the micro BCA Protein Assay Kit (Thermo Scientific) and samples containing equal amounts of protein (5 µg) were resolved by SDS-PAGE and transferred to a PVDF membrane (Invitrogen). Depending on the secondary antibodies (HRP- or IRDye-conjugated) protein bands were detected using either enhanced chemiluminescence reagents (GE Healthcare) and ChemiDoc MO analyzer (Bio-Rad) or Odyssey infrared fluorescence detection system (LI-COR, Lincoln, NE, USA).
The following primary antibodies were used at a 1:500 dilution: CD81 (sc-166029, Santa Cruz Biotechnology), HSP70 (ab181606, Abcam), CD63 (ab59479, Abcam), α-syn (610786, BD Bioscience).
The following primary antibodies were used at a 1:500 dilution: CD81 (sc-166029, Santa Cruz Biotechnology), HSP70 (ab181606, Abcam), CD63 (ab59479, Abcam), α-syn (610786, BD Bioscience).
3
Characterizing Extracellular Vesicle Morphology
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To characterize the morphology of nEVs and CNVs, 10 μl of samples were loaded on the 400 mesh formvar coated copper grid and allowed to incubate for 3 min at room temperature (RT). Samples were drained out with a filter paper and stained with 1% filtered uranyl acetate solution for 1 min. Prepared samples were imaged with Hitachi transmission electron microscopy (TEM) at an acceleration voltage of 100 kV.33 (link) The size distribution and concentration of nEVs and CNVs were detected by Nanosight NS300. Three 30-s videos were taken. Data was analyzed by the build-in algorithm of a NS300 machine and represented as mean ±SD. Classical EV protein biomarkers, including CD81, TSG101, and GAPDH, in nEVs and CNVs were analyzed with western blot. Briefly, 200 μL of nEVs or CNVs lysates were prepared by adding 50 μL of RIPA lysis buffer on ice. Samples were further mixed with 5× loading buffer and placed at 95 °C for 20 min. Gels were running at 60 V for stacking and 100 V for separating. The proteins were transferred to PVDF membrane using Bio-rad mini blotting system at 25 V for 7 min. The membranes were blocked for 1 h in 5% skimmed milk dissolved in TBS. The proteins were detected by incubation with primary antibody conjugated with HRP (CD81: sc-166029, TSG101: sc-7964, GAPDH: sc-32233, Santa Cruz). The membranes were washed thrice prior to imaging.
4
Cardiac Protein Isolation and Analysis
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Proteins were isolated from rat hearts using lysis buffer (KeyGEN BioTECH, China) with a protease and phosphatase inhibitor cocktail solution (KeyGEN BioTECH, China). Thirty micrograms of protein was loaded on a 10% PAGE Gel Fast Preparation Kit (PG112, Epizyme Biotech, China). Proteins were separated and transferred to PVDF membranes (Millipore, USA). Membranes were incubated in TBST skim milk blocking buffer for 1 h at room temperature (RT) followed by primary antibodies overnight at 4 °C. The following primary antibodies were used: CD81 (sc-166,029, Santa Cruz, USA), Alix (sc-53,540, Santa Cruz, USA), MCU (26312-1-AP, Proteintech, USA), MICU1 (ab224161, Abcam, England), NCLX (ab83551, Abcam, England), PINK1 (23274-1-AP, Proteintech, USA), and GAPDH (AF7021, Affinity Biosciences, USA). Membranes were washed with TBST three times, incubated with secondary antibodies (701051, Zen Bioscience, China; ZB-2301, ZSGB-BIO, China) for 1 h at RT and washed with TBST three times. Membranes were probed using a chemiluminescence kit (Millipore, USA) and an Image system (Bio-Rad, USA).
5
Isolation and Western Blot Analysis of Rat Heart Proteins
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Proteins were isolated from rat hearts using lysis buffer (KeyGEN BioTECH, China) with protease and phosphatase inhibitor cocktail solution (KeyGEN BioTECH, China). Thirty micrograms of protein was loaded on a 10% PAGE Gel Fast Preparation Kit (PG112, Epizyme Biotech, China). After Western blotting was performed, PVDF membranes (Millipore, USA) were incubated in TBST skimmed milk blocking buffer for 1 hr at RT and then with primary antibodies overnight at 4℃. Primary antibodies against the following were used: CD81 (sc-166029, Santa Cruz, USA), Alix (sc-53540, Santa Cruz, USA), MCU (26312-1-AP, Proteintech, USA), MICU1 (ab224161, Abcam, England), NCLX (ab83551, Abcam, England), PINK1 (23274-1-AP, Proteintech, USA), and GAPDH (AF7021, A nity Biosciences, USA). After washing with TBST three times, the membranes were incubated with secondary antibodies (701051, Zen Bioscience, China; ZB-2301, ZSGB-BIO, China) for 1 hr at RT and washed with TBST three times again. Membranes were probed with a chemiluminescence kit (Millipore, USA) using an Image system (Bio-Rad, USA).
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