Western blot analysis was performed as previously described (23 (link)). Antibodies against p-RbSer780, Rb, cyclin D1, CDK4 and CDK6, c-Myc, p-AKTSer473, AKT, p-mTORSer2448, mTOR, p-p70S6KThr389, p70S6K, p-AMPKα1Thr172, p-ERK1/2Thr202/Tyr204, ERK1/2, p-PKM2Tyr105, PKM2 were from Cell Signaling Technology, Incorporated (Danvers, MA); anti-p-CDK6Tyr24 and anti-MCT4 were from Santa Cruz Biotechnology, Incorporated (Dallas, TX). Antibodies against CDKN2A/p16INK4a, AMPKα1, GLUT-1 were from Abcam (Cambridge, UK). Antibodies against HIF-1α and HIF-2α were from BD Biosciences (Franklin Lakes, NJ) and Novus Biologicals, LLC (Centennial, CO), respectively. Anti-β-actin (clone B11V08) was from BioVision (Milpitas, CA). Horseradish peroxidase-conjugated secondary antibodies and the chemiluminescence system were from Millipore (Millipore, MA). Reagents for electrophoresis and blotting analysis were from BIO-RAD Laboratories (Hercules, CA). For GLUT-1 detection, cells were lysed in GLUT-1 lysis buffer (1 % Triton X-100, 0.1% SDS, protease inhibitors) for 1 h on ice, and precleared by centrifugation for 10 min at 4°C. Protein extracts were denatured in sample buffer for 30 min before electrophoresis (24 (link)). The chemiluminescent signal was acquired by C-DiGit R Blot Scanner and the bands were quantified by Image Studio™Software, LI-COR Biotechnology (Lincoln, NE).
Chemiluminescence system
Manufactured by Merck Group
Sourced in United States
The Chemiluminescence system is a laboratory equipment designed for the detection and measurement of light-emitting chemical reactions. It provides a reliable and sensitive method for quantifying the presence of specific molecules or analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Image studio software, by LI COR (3 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
P p70s6k thr389, by Cell Signaling Technology (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
P70s6k, by Cell Signaling Technology (2 mentions)
P rb ser780, by Cell Signaling Technology (2 mentions)
Anti β actin clone b11v08, by Abcam (2 mentions)
Anti p cdk6tyr24, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Bca analysis, by Beyotime (2 mentions)
Antibody against hif 1α, by BD (2 mentions)
Anti β actin, by Abcam (2 mentions)
Horseradish peroxidase conjugated protein a, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
36 protocols using chemiluminescence system
1
Western Blot Analysis of Cell Signaling
2
Protein Extraction and Western Blotting
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Protein extraction, solubilization, and protein analysis by Western blotting were performed as described [17 (link)]. A detailed list of the primary selected antibodies is reported in Supplementary Materials (Table S2) . Horseradish peroxidase–conjugated secondary antibodies and chemiluminescence system were from Millipore (Burlington, MA, USA). Reagents for electrophoresis and blotting analysis were from BIO-RAD Laboratories (Hercules, CA, USA). The chemiluminescent signal was acquired by C-DiGit®. Blot Scanner and the spots were quantified by Image StudioTM Software, LI-COR Biotechnology (Lincoln, NE, USA).
3
Extraction and Detection of HIF-1α Protein
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For the extraction of the total protein, the cultivated cells were lysed using the lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodium 125 deoxycholate, 0.1% SDS, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM Na3VO4, and inhibitor cocktail tablets]. A designated equal amount of the total proteins extracted from different treatments was resolved on a sodium dodecyl sulfate-polyacrylamide gel (i.e., 7.5%, 12.5% polyacrylamide gels) electrophoresis (SDS-PAGE) and transferred onto the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the primary rabbit monoclonal anti-HIF-1α from Life Technology Invitrogen (Thermo Fisher Scientific Corp., Waltham, MA, USA). Then, they were overlaid with the secondary polyclonal goat anti-rabbit antibodies (Abcam, Cambridge, UK). The protein bands were visualized using a chemiluminescence system (Millipore, Burlington, MA, USA).
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For the extraction of the total protein, the cultivated cells were lysed using the lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodium 125 deoxycholate, 0.1% SDS, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM Na3VO4, and inhibitor cocktail tablets]. A designated equal amount of the total proteins extracted from different treatments was resolved on a sodium dodecyl sulfate-polyacrylamide gel (i.e., 7.5%, 12.5% polyacrylamide gels) electrophoresis (SDS-PAGE) and transferred onto the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the primary rabbit monoclonal anti-HIF-1α from Life Technology Invitrogen (Thermo Fisher Scientific Corp., Waltham, MA, USA). Then, they were overlaid with the secondary polyclonal goat anti-rabbit antibodies (Abcam, Cambridge, UK). The protein bands were visualized using a chemiluminescence system (Millipore, Burlington, MA, USA).
4
Western Blot Analysis of Protein Expression
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The treated cells were washed twice with PBS and placed on ice with RIPA lysate for at least 30 min. The total protein of the cells was extracted and quantified. Cell lysates were added to 10% or 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for separation and transferred to NC membranes, which were then sealed with 5% skim milk and incubated overnight with primary antibody at 4°C. The membrane was washed the next day with TBST and incubated with a secondary antibody for 1 hour at room temperature. Protein expression was detected by a chemiluminescence system (Millipore) according to the instructions.
5
Western Blot Analysis Protocol
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WB analysis was performed as described previously12 (link). The antibodies against PCNA(Cell Signaling, 13110), HMGB1(Cell Signaling, 6893), LC3B(Cell Signaling, 12741), p62(Cell Signaling, 5114), Becline(Cell Signaling, 3495), BAX(abcam, ab32503), BCL2(Proteintech, 12789-1-AP), Cas8(abcam, ab25901), PARP1(Proteintech, 13371-1-AP), AKT(Cell Signaling, 9272), p-AKT(ser473) (Cell Signaling, 4060), p-AKT(thr308) (Cell Signaling, 13038), p-PTEN(ser380)(Cell Signaling, 9551), p-GSK-3β(ser9) (Cell Signaling, 5558), p-c-Raf(ser259) (Cell Signaling, 9421), mTOR(Cell Signaling, 2983), p-mTOR(ser2448) (Cell Signaling, 5536), p27(abcam, ab32034), p-p27(s10)(abcam, ab62364), CDK2(abcam, ab32147), CDK4(Santa Cruz, sc-23896), CyclinB1(abcam, ab181593), CyclinD1(abcam, ab134175), Cyclin E(Santa Cruz, sc-377100), NEDD4 L(Santa Cruz, sc-514954), C/EBPα(Cell Signaling, 8178) and GAPDH(Cell Signaling, 5174) were diluted in TBS containing 5% milk and 0.1% Tween 20. Signals were detected using a chemiluminescence system (Millipore, Bedford, MA, USA).
6
Quantitative Protein Analysis by Western Blot
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Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extracts were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After incubation with a high-affinity anti-KRT6B antibody (1:2000, Proteintech, USA), anti-vimentin antibody (1:1000, Proteintech, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washing, the signals were detected using a chemiluminescence system (Millipore, USA) and analysed using Image Lab Software (Bio-Rad, USA).
7
Quantitative Protein Expression Analysis
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Total proteins extracted from cells or tumor tissues were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins in the gel were electrotransferred onto polyvinylidene fluoride membranes. The proteins were then probed with antibodies against DR6 (BioVision, Milpitas, CA, USA), IL-6R (Proteintech, Rosemont, IL, USA), VEGF-R-2 (Abcam, Cambridge, MA, USA), VEGF-D and PDGFR-α (Biogot, Nanjing, China), caspase 3, caspase 6, p-IκBα, p-P38, P38, p-STAT3 or STAT3 (Cell Signaling Technology, Danvers, MA, USA) and then incubated with an appropriate horse radish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology). The target proteins were visualized with a chemiluminescence system (Millipore, MA, USA) and exposed to X-ray film. The blot images were scanned and processed using Microsoft Office Picture Manager. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (KangChen Biotech, Beijing, China) was used as an internal control.
8
Western Blotting Analyses of Cell Signaling Pathways
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Analyses of western blotting were performed as previously described [19 (link)].
Antibodies against p-RbSer780 (#9307), Rb (#9309), cyclin D1 (#2926), CDK6 (#3136), c-myc (#9402), p-AKTSer473 (#9271), AKT (#9272), p-mTORSer2448 (#2971), mTOR (#2972), p-ERK1/2Thr202/Tyr204 (#4370), ERK1/2 (#4695), were from CST (Danvers, MA); anti-p-CDK6Tyr24 (sc-293,097) was from Santa Cruz Biotechnology, Incorporated (Dallas, TX). Anti CDKN2A/p16INK4a (ab81278) and anti-GLUT-1 (ab40084) were from Abcam (Cambridge, UK). Antibody against HIF-1α (#610959) was from BD Biosciences (Franklin Lakes, NJ). Anti-β-actin (#3598) was from BioVision (Milpitas, CA). All the antibodies were used at the recommended dilution of 1:1000. Horseradish peroxidase-conjugated secondary antibodies (1:10,000) and chemiluminescence system were from Millipore (Millipore, MA). Reagents for electrophoresis and blotting analysis were from BIO-RAD Laboratories (Hercules, CA).
Antibodies against p-RbSer780 (#9307), Rb (#9309), cyclin D1 (#2926), CDK6 (#3136), c-myc (#9402), p-AKTSer473 (#9271), AKT (#9272), p-mTORSer2448 (#2971), mTOR (#2972), p-ERK1/2Thr202/Tyr204 (#4370), ERK1/2 (#4695), were from CST (Danvers, MA); anti-p-CDK6Tyr24 (sc-293,097) was from Santa Cruz Biotechnology, Incorporated (Dallas, TX). Anti CDKN2A/p16INK4a (ab81278) and anti-GLUT-1 (ab40084) were from Abcam (Cambridge, UK). Antibody against HIF-1α (#610959) was from BD Biosciences (Franklin Lakes, NJ). Anti-β-actin (#3598) was from BioVision (Milpitas, CA). All the antibodies were used at the recommended dilution of 1:1000. Horseradish peroxidase-conjugated secondary antibodies (1:10,000) and chemiluminescence system were from Millipore (Millipore, MA). Reagents for electrophoresis and blotting analysis were from BIO-RAD Laboratories (Hercules, CA).
9
Protein Expression Analysis by Western Blot
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The cells were lysed by lysis buffer on ice with protease inhibitors (Roche, Penzberg, Germany). The proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore). Then, the membranes were blocked with 5% skim milk and incubated with rabbit anti-human antibodies against TIMP-2, MMP-2, or MMP-9 (Santa Cruz Biotechnology) or GAPDH (Cell Signaling) overnight at 4°C. The membranes were hybridized with a goat anti-rabbit secondary antibody for 1 h at room temperature. Specific binding was detected using a chemiluminescence system (Millipore).
10
Quantifying GPRC5A Protein Expression
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Protein was isolated from the cells and tissues with RIPA lysis buffer containing 1% protease inhibitor cocktails (Pierce Biotechnology, Inc.; Thermo Fisher Scientific, Inc.). After sample buffer was added to the proteins (each well, 30 µg per sample), proteins were boiled at 95°C for 10 min. Then, the proteins were separated using 10% polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes with 100 V transfer-molded voltage lasting for 45 to 70 min. After determination of the protein concentration, primary antibodies for western blotting were applied which included anti-GPRC5A (dilution, 1:1,000; PAB14597; Abnova, Taipei, Taiwan) and anti-GAPDH (dilution, 1:2,000; ab8245; Abcam, Cambridge, UK). HRP-conjugated IgG (1:5,000) antibody was used as the secondary antibody. After which membranes were washed 3 times (5 min/time). Development was completed with chemiluminescence reagents. GAPDH was used as an internal reference. Bands were visualized with a Bio-Rad Gel Doc EZ imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The specific bands were visualized with a chemiluminescence system (Millipore), and then visualized with Quantity One software 4.6.2 (Bio-Rad Laboratories, Inc.).
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