Secreted factors in tissue culture supernatants and sera were quantified by ELISA according to the manufacturer’s instructions (eBioscience), or by using mulitplex bead capture assays and a Bio-Plex 200 analyzer (Bio-Rad). Cell lysates were resolved on SDS-PAGE gels, probed with primary Abs, and immunoreactive proteins were visualized with HRP-coupled secondary Abs and chemiluminescence reagents (Bio-Rad, Pierce, or Cell Signaling Technology). Blots were visualized using the ChemiDoc MP imaging system and in some cases were quantified using Image Lab software (Bio-Rad).
Chemiluminescence reagent
Manufactured by Bio-Rad
Sourced in United States
The Chemiluminescence Reagent is a laboratory product that emits light as a result of a chemical reaction. It is used to detect and quantify the presence of specific molecules in a sample. The reagent is designed to provide a sensitive and reliable method for various analytical applications, including immunoassays and gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (5 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Autoradiography film, by Genesee Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bio plex 200 analyzer, by Bio-Rad (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (2 mentions)
Goat anti rabbit β actin antibody, by Cell Signaling Technology (2 mentions)
Chemidoc imaging machine, by Bio-Rad (2 mentions)
Coomassie protein assay reagent, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (2 mentions)
Ripa cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
40 protocols using chemiluminescence reagent
1
Quantification of Secreted Factors
2
Western Blot and qRT-PCR Analysis of Cardiac Proteins
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We isolated the total proteins in the cardiomyocytes
or heart tissue and conducted protein electrophoresis
by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). The protein was
transferred to immobilized membranes (Millipore,
Billerica, MA, USA), which were incubated with
the corresponding primary antibodies that included
total (T) P38 (1:1000 dilution, #ab178867) and
phosphorylated (P) P38 (1:1000 dilution, Abcam
#ab4822), NADPH oxidase 4 (NOX4, 1:1000 dilution, #ab154244) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, 1:1000 dilution, Cell
Signalling Technology, #5174). After incubation with
the secondary antibody, the blots were developed with
enhanced chemiluminescence reagents (Bio-Rad,
Hercules, CA, USA) and captured by a ChemiDoc MP
Imaging System (Bio-Rad). GAPDH was used as an
internal reference protein.
Total RNA from mouse heart tissue or cardiomyocytes was extracted. We reverse transcribed
2 μg of mRNA into cDNA with a cDNA Synthesis Kit (Roche Diagnostics). A Light cycler 480
instrument (software version 1.5, Roche) and SYBR Green PCR premix (Roche) were used for
the amplification reaction. GAPDH was used as an internal reference gene
to standardize and quantify gene expressions.
or heart tissue and conducted protein electrophoresis
by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). The protein was
transferred to immobilized membranes (Millipore,
Billerica, MA, USA), which were incubated with
the corresponding primary antibodies that included
total (T) P38 (1:1000 dilution, #ab178867) and
phosphorylated (P) P38 (1:1000 dilution, Abcam
#ab4822), NADPH oxidase 4 (NOX4, 1:1000 dilution, #ab154244) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, 1:1000 dilution, Cell
Signalling Technology, #5174). After incubation with
the secondary antibody, the blots were developed with
enhanced chemiluminescence reagents (Bio-Rad,
Hercules, CA, USA) and captured by a ChemiDoc MP
Imaging System (Bio-Rad). GAPDH was used as an
internal reference protein.
Total RNA from mouse heart tissue or cardiomyocytes was extracted. We reverse transcribed
2 μg of mRNA into cDNA with a cDNA Synthesis Kit (Roche Diagnostics). A Light cycler 480
instrument (software version 1.5, Roche) and SYBR Green PCR premix (Roche) were used for
the amplification reaction. GAPDH was used as an internal reference gene
to standardize and quantify gene expressions.
3
Western Blot Analysis of Leukemia Cells
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Western blot analysis was performed using standard techniques. Briefly, transiently transfected leukemia cells were harvested and lysed by RIPA buffer (Thermo Scientific, Waltham, MA, USA). Proteins (40 μg) from the lysate were fractionated by electrophoresis through polyacrylamide gels (Bio-Rad, Richmond, CA, USA) and transferred to PVDF membranes. Blots were incubated with primary antibodies over night at 4 °C and incubated with second HRP-conjugated antibody for 1 h. Signals were measured by chemiluminescence reagents (Bio-Rad) with imaging system (Bio-Rad). The following antibody was used: HOXB3 (ab82945, Abcam, Cambridge, MA, USA); CDCA3 (ab166902, Abcam); DNMT3B (ab176166, Abcam). As necessary, blots were stripped and reprobed with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, US) as an endogenous control.
4
Oligonucleotide Primers and Immunoblot Analysis
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Oligonucleotide primers for convention PCR was synthesized by Integrated DNA technologies (Coralville, Iowa) and are listed in Table S1 . Total RNA and proteins were isolated from various using standard protocols as we described11 (link). Total RNA was subjected to conventional PCR analyses using a master mix from Bio-Rad Laboratories (Hercules, CA). Protein extracts were subjected to immunoblot analysis using antibodies against PD-L1, EGFR, Alix, p-ERK, ERK, p-JNK, JNK, p-p38, p38, cyclin D1 (Cell Signaling Technology, Danvers, MA, USA), CD63, Her2 and GAPDH (Santa Cruz Biotechnology, Dallas, TX), Rab27a (Proteintech, Chicago, IL, USA) and nSMase (Abcam, San Francisco, CA, USA). Immune complexes were detected with appropriate secondary antibodies from Jackson ImminoResearch Inc. (West Grove, PA, USA) and chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) as described13 (link),44 (link). Immunoblot signals were captured using the Image Quant Las 300 (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/ ).
5
Protein Expression Analysis Protocol
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Radioimmunoprecipitation assay lysis buffer (Sigma) was adopted to obtain protein samples. Forty micrograms of protein samples were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After sealing for 1 h in 5% milk, primary antibodies were incubated with the membrane, including anti-Cyclin D1 (ab40754; Abcam, Cambridge, MA, USA), anti-caspase 3 (ab13585; Abcam), anti-matrix metallopeptidase 2 (anti-MMP2; ab181286; Abcam), anti- matrix metallopeptidase 2 (anti-MMP9; ab137867; Abcam), anti-GOT2 (SAB2100950; Sigma), anti-proliferating cell nuclear antigen (anti-PCNA; ab29; Abcam), and anti-β-Actin (ab8226; Abcam). After washing using Tris buffered saline–Tween 20 (Sangon Biotech) for 3 times, secondary antibody (Abcam) was added to mark the primary antibodies. Protein bands were determined using chemiluminescence reagents (Bio-Rad).
6
Western Blot Protein Detection
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Cell pellets were resuspended in lysis solution (Beyotime, Shanghai, China) supplemented with protease inhibitors (Biotool, Houston, TX, USA). Samples were separated on 10% SDS-PAGE gel and then transferred to 0.22 μm PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then immersed in a 5% milk solution for 60 min and exposed to primary antibodies for 16 h at 4 °C before adding HRP-conjugated secondary antibodies for 1 h at room temperature. After the membranes were washed, chemiluminescence reagents (Yeason Shanghai, China) were added and detection was performed using the Chemi Doc Imaging System (Bio-Rad, Hercules, CA, USA). Details of the antibodies used are shown in Supplementary Table S1 .
7
SDS-PAGE and Western Blot Analysis
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mAbs and their fragments were fractionated by 12.5% SDS–PAGE. The gels were then either stained with Coomassie Brilliant Blue R-250 or blotted on nitrocellulose membranes (GE Healthcare). After blocking for 1 h in PBS containing 3% BSA, the membranes were incubated with anti-human IgG (γ-chain-specific; Sigma–Aldrich) or anti-human IgG (CH + CL-specific; Promega, Madison, WI) conjugated to horseradish peroxidase (HRP) at a concentration of 0.2–0.5 μg/mL PBS containing 0.05% Tween 20 (PBST) and 0.5% BSA for 90 min prior to development using chemiluminescence reagents (Bio-Rad). Monoclonal anti-KDEL antibodies (Merck Millipore, Darmstadt, Germany) were used at a concentration of 0.2 μg/mL in PBST containing 3% BSA and detected with anti-mouse IgG-HRP (Jackson ImmunoResearch, West Grove, PA) as outlined above.
8
Evaluating Protein Signaling Pathways
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Western blot analysis was performed as we previously reported [30 (link)]. Cells were seeded in 6-well plates. After 24 h, the cells in each well were treated with HCQ or Spautin-1 for 1 h and then BKM120 for 48 h. Total proteins were separated and blotted. The signal was detected by a ChemiDocXRS+ System (BIO-RAD) after exposure to chemiluminescence reagents (BIO-RAD). β-actin served as the loading control.
9
Rhotekin-RBD Pull-Down Assay for RhoA
10
Western Blot Analysis of Cellular Protein Expression
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Western blots were performed as previously described.[35 ] Cells were seeded in six‐well plates at a density of 2 × 105 cells per well in 2 mL of medium. After 24 h, the cells in each well were treated with STELB and/or IR or TMZ. Cells were lysed, and total proteins were harvested. Equal amounts of protein (20–50 µg) were separated using 8% or 12% SDS‐PAGE and transferred to polyvinylidene difluoride membranes (Bio‐Rad). After blocking in 5% nonfat dry milk, the membranes were incubated with appropriate primary antibodies overnight at 4 °C, washed, and incubated with respective HRP‐conjugated secondary antibodies for 1 h at room temperature. The signals were detected using a ChemiDoc XRS+ System (Bio‐Rad) after exposure to chemiluminescence reagents (Bio‐Rad), and β‐actin served as the loading control.
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