Western blot was conducted as previously described24 (link). Briefly, protein samples were prepared with RIPA lysis buffer (Sigma-Aldrich, Cambridge, MA, USA) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific). After separation by sodium dodecyl sulfate polyacrylamide gel, proteins (30 μg) were transferred to a PVDF membrane (Millipore, Boston, MA, USA). After blocking with 5% nonfat milk, the PVDF membrane was incubated with primary antibody in a cold room overnight. After three washes with Tris-buffered saline Tween (TBST) for 15 min, the PVDF membrane was incubated with peroxidase-conjugated secondary antibody. Then after three washes with TBST for 15 min, the protein was visualized with ECL detection solution (Millipore). The primary antibodies were listed as follows: anti-CISD2, anti-GAPDH, anti-GSK3β, anti-p-GSK3β, anti-vimentin, and anti-N-cadherin (purchased from Sigma-Aldrich, St. Louis, MO, USA); anti-β-catenin, anti-p-β-catenin, anti-c-Myc, anti-E-cadherin, and anti-γ-catenin (purchased from Abcam, Cambridge, UK). GAPDH was used as internal control. The intensity of the protein bands was quantified with ImageJ and normalized to that of GAPDH.
Ecl detection solution
Manufactured by Merck Group
Sourced in Denmark
The ECL detection solution is a laboratory product designed for enhanced chemiluminescence (ECL) detection, a technique used in various analytical and research applications. The solution contains the necessary components to facilitate the chemiluminescent reaction, enabling the detection and visualization of target molecules or analytes. The core function of this product is to provide a reliable and sensitive means of detection without further interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Peroxidase conjugated secondary antibody, by Agilent Technologies (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Spectrophotometric plate reader, by Tecan (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti cisd2, by Merck Group (1 mentions)
Anti gsk3β, by Merck Group (1 mentions)
Anti p gsk3β, by Merck Group (1 mentions)
Anti n cadherin, by Merck Group (1 mentions)
Anti p β catenin, by Abcam (1 mentions)
Anti γ catenin, by Abcam (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti β catenin, by Abcam (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Smurf1, by Abcam (1 mentions)
7 protocols using ecl detection solution
1
Comprehensive Western Blot Profiling
2
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific, USA) for extracting total proteins. The protein concentrations were measured by a BCA protein assay kit (Transgen Biotech, China), separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The membranes were then blocked by 10% nonfat milk and incubated with the corresponding antibody (CKIP-1, Akt, p-Akt, Gsk3β, β-catenin, p-β-catenin, Smurf1, and GAPDH; Abcam, Cambridge, MA, USA) diluted according to the instructions for 12 h at 4°C. After washing 3 times with PBS/0.1% Tween-20, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG and anti-rabbit IgG at 1:10000 dilution; Abcam). Protein expression levels were detected with ECL detection solution (Millipore, USA) and visualized with chemiluminescence detection system. The bands signal were analyzed using a gel imaging system (Bio-Rad).
3
Ischemic Cortex Separation and Protein Analysis
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Used a similar approach for separating the ischemic cortex,51 (link),52 (link) a 4mm thick coronal block was cut from the mouse brain, which containing infarct core area (has color difference with surrounding tissues). In ischemic side, the cortex at an angle of about -60 to 30 degrees horizontally from the center of the block was separated as infarct cortex. Cells or brain tissues were homogenized in ice-cold IP lysis buffer (Beyotime) containing protease and phosphatase inhibitors. The protein concentration was determined using the BCA protein assay kit. Protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to nitrocellulose membrane (0.22 μm). After blocking with 3 % BSA for about 2 h, the membrane was incubated with the indicated primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies. The membrane was washed three times and detected by ECL detection solution (Millipore). The bands were visualized in an automatic chemiluminescence imaging system (Protein Simple, Pluorchem E) and analyzed with Image J software.
4
Western Blot Analysis of ER Stress and Autophagy
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Total protein samples were separated on 10% or 8% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk or 5% bovine serum albumin for 1 h at room temperature. Then, the membranes and primary antibodies were incubated overnight at 4 ℃. The primary antibodies included GRP78 (Cell Signaling Technology, Beverly, MA, USA), PERK (Cell Signaling Technology), IRE1 (Cell Signaling Technology), ATF6 (Santa Cruz, Dallas, TX, USA), p-JNK (Cell Signaling Technology), p-PI3K (Cell Signaling Technology), p-AKT (Cell Signaling Technology), LC3B (Cell Signaling Technology), and p62 (Abcam, Cambridge, UK). β-actin (Abcam) was chosen as an endogenous control. Next, the PVDF membranes were incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing with TBST three times, the proteins were visualized with ECL detection solution (Millipore) using automatic gel imaging analysis system.
5
Quantifying ER Expression in Breast Cancer Cells
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After 48 hour incubation of untreated and transfected MCF-7 and MDA-MB-231 cells, total RNA and protein were extracted using peqGOLD TriFast (Peqlab, Erlangen, Germany) according to the manufacturer`s instructions.
To determine mRNA expression of ER, a total of 200 ng RNA from basal and transfected cells was reverse transcribed using the First strand cDNA synthesis kit (Thermo Scientific). The mRNA expression levels were subsequently quantified by real-time PCR using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) and the following primers for ER (forward: 5′-GCATTCTACAGGCCAAATTCA-3′ and reverse: 5′-TCCTTGGCAGATTCCATAGC-3′). GAPDH (forward: 5′-CCTGCACCACCAACTGCTTAG-3′ and reverse: 5′-TGGCATGGACTGTGGTCATG-3′) served as reference gene. Protein levels of ER in basal and transfected MCF-7 cells were investigated by Western blot analysis. Thirty μg of cell lysates were electrophoretically separated and blotted onto a PVDF membrane (Millipore, Billerica, USA) which was subsequently incubated with antibodies specific for ER (Thermo Scientific) and GAPDH (Santa Cruz, Heidelberg, Germany) overnight. Detection of the proteins was carried out using peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and the chemiluminescence ECL detection solution (Sigma-Aldrich St.Louis, Missouri, USA).
To determine mRNA expression of ER, a total of 200 ng RNA from basal and transfected cells was reverse transcribed using the First strand cDNA synthesis kit (Thermo Scientific). The mRNA expression levels were subsequently quantified by real-time PCR using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) and the following primers for ER (forward: 5′-GCATTCTACAGGCCAAATTCA-3′ and reverse: 5′-TCCTTGGCAGATTCCATAGC-3′). GAPDH (forward: 5′-CCTGCACCACCAACTGCTTAG-3′ and reverse: 5′-TGGCATGGACTGTGGTCATG-3′) served as reference gene. Protein levels of ER in basal and transfected MCF-7 cells were investigated by Western blot analysis. Thirty μg of cell lysates were electrophoretically separated and blotted onto a PVDF membrane (Millipore, Billerica, USA) which was subsequently incubated with antibodies specific for ER (Thermo Scientific) and GAPDH (Santa Cruz, Heidelberg, Germany) overnight. Detection of the proteins was carried out using peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and the chemiluminescence ECL detection solution (Sigma-Aldrich St.Louis, Missouri, USA).
6
Quantifying Exosomal Proteins for Western Blot
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To calculate the adequate protein amounts for carrying out a Western blot, the protein concentrations were at first measured with the DC Protein Assay Kit (BioRad, Munich, Germany) at a wavelength of 650 nm on a spectrophotometric plate reader (Tecan). A standard curve of 0, 0.625, 1.25, 2.5, 5, and 10 mg/ml BSA (bovine serum albumin; Sigma Aldrich Chemie, Munich, Germany) was applied by the double-dilution method. Five microliters of exosomes, exosome supernatant, and BSA (Sigma Aldrich Chemie) standard protein samples, all solved in RIPA buffer (Merck, Darmstadt, Germany), were added to 96-well plates according to the manufacturer’s instructions. The protein concentrations were then calculated according to a linear equation by applying the regression method.
Exosomes were lysed in RIPA buffer (Merck) and PBS (Life Technologies), and 30 μg of proteins from exosomes and exosome supernatant were electrophoretically separated and blotted onto a PVDF membrane (Millipore, Billerica, USA) which was subsequently incubated with antibodies specific for CD63 (ABGENT, San Diego, California, USA), CD81 (Invitrogen, Darmstadt, Germany) and AGO2 (TAKARA BIO INC, Shiga, Japan) overnight. Detection of the proteins was carried out using peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and the chemiluminescence ECL detection solution (Sigma-Aldrich, St. Louis, MO, USA).
Exosomes were lysed in RIPA buffer (Merck) and PBS (Life Technologies), and 30 μg of proteins from exosomes and exosome supernatant were electrophoretically separated and blotted onto a PVDF membrane (Millipore, Billerica, USA) which was subsequently incubated with antibodies specific for CD63 (ABGENT, San Diego, California, USA), CD81 (Invitrogen, Darmstadt, Germany) and AGO2 (TAKARA BIO INC, Shiga, Japan) overnight. Detection of the proteins was carried out using peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and the chemiluminescence ECL detection solution (Sigma-Aldrich, St. Louis, MO, USA).
7
Exosome Protein Quantification and Detection
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The protein concentrations were measured with the DC Protein Assay Kit (BioRad, Munich, Germany) at a wavelength of 650 nm on a spectrophotometric plate reader (Tecan). A standard curve of 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, and 10 mg/mL bovine serum albumin (BSA; Sigma–Aldrich Chemie, Munich, Germany) was applied by the double-dilution method. Then, 2.5 μL of exosomes and BSA standard samples, all resuspended in Phosphate-Buffered Saline (PBS) buffer (Life Technologies, Paisley, UK) were added to 96-well plates according to the manufacturer’s instructions. The protein concentrations were calculated according to a linear equation of y = mx + n by applying the linear regression method. For Western blot, 30 μg of exosomes resuspended in PBS buffer (Life Technologies) were electrophoretically separated and blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) which was subsequently incubated with an antibody specific for the exosomal marker CD63 (Abgent, San Diego, CA, USA) overnight. Detection of the proteins was carried out using a peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark) and a chemiluminescence ECL detection solution (Sigma–Aldrich, St. Louis, MO, USA).
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