Electrochemiluminescence kit

Cell lysates were prepared at 75% of confluence by using 500 μL of radio-immunoprecipitation assay buffer (25 mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) in 10-cm culture dishes with a 20-minute incubation on ice. The protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Hercules, CA). Immunoblot analyses were performed as previously described [37 (link), 38 (link)]. β-Actin antibodies were obtained from Santa Cruz Biotechnology. HER2, Claudin-1, ZEB1, ZO-1, Fak, pFak, Smad, pSmad were purchased from Cell Signaling Technology. The secondary antibodies were the F(ab)₂ fragment of donkey anti-mouse immunoglobulin (product NA931) or of donkey anti-rabbit immunoglobulin (product NA9340) linked to horseradish peroxidase from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The immunoblot reagents were from an electrochemiluminescence kit (Amersham Biosciences, NJ, USA).

For cell/EV lysis, the harvested cells/EVs were incubated on ice in a whole-cell-extract lysis buffer and centrifuged, and the protein concentration was then measured by a Bradford assay (Biorad Laboratories, CA, USA). For the Western blot analysis, the lysates were then boiled with sample buffer before being separated on SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA) and blocked with 5% nonfat milk/TBST buffer. Using an electrochemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA), we detected the binding of specific antibodies. The EV marker-specific antibody CD63 was purchased from Santa Cruz Biotechnology, while the ER marker GRP78-specific antibody was purchased from Abcam (Cambridge, UK). To investigate changes in the activity of downstream EGFR signaling after treatment, the following antibodies were used: (1) anti-phospho-Stat3 (Tyr705) (Cell Signaling, Danvers, MA, USA), (2) anti-Stat3 (BD Biosciences, San Jose, CA, USA), (3) anti-actin (Millipore), (4) anti-phospho-Akt (Ser473) (Cell Signaling), (5) anti-Akt (Cell Signaling), (6) anti-phospho-Erk (R&D, Minneapolis, MN, USA), (7) anti-Erk (Santa Cruz Biotechnology, Santa Cruz, CA, USA), (8) anti-E-cadherin (Cell Signaling), and (9) anti-Zeb1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

To analyze protein expression in cells, immunoblotting analysis was performed as previously described [2 (link)]. Antibodies against the following proteins were obtained from Santa Cruz Technology (California, US): Aur A (sc-25425), Aur B (sc-25426), BRCA1 (sc-6954), CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), cyclin D1 (sc-718), cyclin E (sc-247), cyclin A (SC-751). Antibodies against BRCA2 (19791-1-AP) and cyclin B1 (cs-4135) were obtained from Proteintech (Chicago, USA) and Cell Signaling Technology (Massachusetts, US), respectively. The detection of β-actin (A2228, Sigma Aldrich, St. Louis, MO) was used as a loading control. The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or of donkey anti-rabbit immunoglobulin linked to horseradish peroxidase from Cell Signaling Technology (Massachusetts, US). Immunoblotting reagents were from an electrochemiluminescence kit (Amersham Biosciences). To test whether the expression levels of proteins were regulated through proteasome-mediated degradation, cells were exposed with 20 μM MG132 (#S2619, Selleck Company, Texas, America) for 3 h and then harvested for Western blotting analysis.

Antibodies against LC3B (3868S), Ub (3936S), and active Caspase-3 (9664S) were purchased from Cell Signaling. Antibody against β-actin (sc-8432) was obtained from Santa Cruz. Antibody against RACK1 (610178) was from BD Biosciences. Antibody against electron-transferring-flavoprotein dehydrogenase (ETFDH) (A6585) was from ABclonal Biotech. Antibody against very long-chain acyl-CoA dehydrogenase (VLCAD) (14527-1-AP) was from Proteintech. Total protein extracts were prepared and dissolved in SDS sample buffer. Protein extracts were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were probed with antibodies and visualized with an electrochemiluminescence kit (Amersham).

To analyze protein expression in cells, cell lysates were prepared at 75% of confluence by using 500 μL of radioimmunoprecipitation assay buffer (25 mM Tris—HCl at pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) in 10-cm culture dish, with a 20-minute incubation on ice. Protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Hercules, CA). Immunoblot analyses were performed as previously described[29 (link)]. STC2 and β-actin antibodies were obtained from Santa Cruz Biotechnology. pPKC, Claudin-1, MMP9, Slug, Twist, Vimentin, ZEB1,ZO-1 were purchased from Cell Signaling Technology. The secondary antibodies were F(ab)2 fragment of donkey anti-mouse immunoglobulin (product NA931) or of donkey anti-rabbit immunoglobulin (product NA9340) linked to horseradish peroxidase from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). Immunoblot reagents were from an electrochemiluminescence kit (Amersham Biosciences).

To determine the mechanism of MgT in neuroprotection during hypoxia, we measured the expression of a glutamate transporter, excitatory amino acid transporter (EAAT) 4, by western blot. Following behavioral testing, zebrafish brains were homogenized in a lysis buffer (radio-immunoprecipitation buffer; Sigma) containing a protease inhibitor cocktail (Roche). Protein concentration was determined using the Bradford method. Proteins (20 μg) were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to membranes. The membranes were blocked with 5% skim milk in 1 × tris-buffered saline (TBS) at room temperature for 1 hour. The membranes were then incubated with a rabbit anti-EAAT4 antibody (ab41650, Abcam, Cambridge, MA) or rabbit anti-beta actin (A5441, Sigma-Aldrich) overnight at 4 °C. Membranes were washed three times in 1 × TBS + 0.05% Tween 20 and incubated with a 1:2000 (EAAT4) or 1:5000 (beta actin) dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody. An electro-chemiluminescence kit was used to develop the western blots (Amersham, Boston, MA, USA). Quantitative analysis of densitometry was performed using ImageJ (v. 1.52a, National Institutes of Health, USA).