Western blotting was performed on cultured cells or tissue samples after the indicated treatments. Cell lysates were collected using a sodium dodecyl sulfate lysis buffer (Beyotime Biotechnology, Shanghai, China). Equal amounts of total protein (approximately 15 μg for cell samples and 60 μg for tissue samples) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% nonfat powdered milk (Sangon, Shanghai, China) in TBS with Tween-20 (TBST) at about 20°C for 1.5 h, incubated with primary antibodies overnight at 4°C, washed three times with TBST, and then incubated with secondary antibodies for 1 h. The signal intensity of protein bands was visualized using an Enhanced Chemiluminescence Detection Kit (Tanon, Shanghai, China). A semi-quantitative evaluation of protein density was performed using ImageJ (Version 1.5.3). The following antibodies were used at a 1:1000 dilution: anti-GGH (Cat. #138495; Abcam, Cambridge, UK), anti-PKM (Cat. #150377; Abcam), anti-GLUT1 (Cat. #115730; Abcam), anti-LDHA (Cat. #52488; Abcam), anti-β-actin (Cat. #4970, Abcam), and anti-GAPDH (Cat. #2118; Cell Signaling, Danvers, MA, USA).
Sodium dodecyl sulfate lysis buffer
Manufactured by Beyotime
Sourced in China
Sodium dodecyl sulfate (SDS) lysis buffer is a solution used to disrupt and lyse cells, solubilize proteins, and denature nucleic acids. It is a commonly used reagent in various biological and biochemical applications, such as protein extraction, Western blotting, and cell lysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti pkm, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Non fat powdered milk, by Sangon (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti glut1, by Abcam (1 mentions)
Anti ldha, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Twist1, by Abcam (1 mentions)
Peroxidase conjugated anti igg, by Abcam (1 mentions)
Pierce ecl substrate western blot detection system, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
Gapdh, by GeneTex (1 mentions)
Vimentin, by GeneTex (1 mentions)
N cadherin, by GeneTex (1 mentions)
Enhanced bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
0.22 μm polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using sodium dodecyl sulfate lysis buffer
1
Protein Expression Analysis by Western Blotting
2
Western Blot Analysis of EMT Markers
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Tissue samples were ground to powder in liquid nitrogen. Total proteins were extracted from tissue powder or treated cells using sodium dodecyl sulfate lysis buffer (Beyotime, Shanghai, People’s Republic of China) for 30 minutes at 4°C, and an equal amount of protein was separated using 10% polyacrylamide sodium dodecyl sulfate gels. Then, the proteins were transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific) and were probed with primary antibodies against E-cadherin (Abcam, Cambridge, UK), vimentin (GeneTex, San Antonio, TX, USA), TWIST1 (Abcam), N-cadherin (GeneTex), matrix metalloproteinase (MMP)-2 (Abcam), MMP-9 (Abcam), or GAPDH (Gene-Tex). The membranes were incubated overnight at 4°C, followed by incubation with secondary antibody peroxidase-conjugated anti-IgG (Abcam), and detected using a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system; Thermo Fisher Scientific). Quantity One 4.5.0 software (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to quantify the integrated density of the protein bands.
3
Protein Extraction and Quantification
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The cultured cells or clinical samples were lysed and ultrasonicated with sodium dodecyl sulfate lysis buffer (Beyotime, Shanghai, China) on ice for 5 min and denaturalized in a water bath at 100℃ for 5 min. The supernatant was then transferred into new Eppendorf tubes and used to determine the resultant protein concentration using the Enhanced Bicinchoninic Acid Protein Assay Kit (Beyotime).
4
Immunoblotting Analysis of HNSCC-derived sEVs and Lysates
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The CR cells/HNSCC/BMSC cells‐derived sEVs and cell lysates from M2 were collected in sodium dodecyl sulfate lysis buffer (Beyotime, Shanghai, China) and stored at ‐80°C until use. The cell lysates (20 μg) and sEVs proteins (3 μg), were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred onto 0.22‐μm polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked with nonfat milk for 2 h at room temperature and incubated overnight at 4°C with primary antibodies. β‐Actin and GAPDH were used as loading controls. The membranes were treated with secondary antibodies and visualized using ECLUltra (New Cell and Molecular Biotech, Suzhou, China). The antibodies used are listed in Table S2 .
5
Immunoblotting Analysis of HIF-1α in Cells
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Immunoblotting analysis of proteins in HT-29 cells and RAW 264.7 cells was performed in the lysis buffer containing sodium dodecyl sulfate lysis buffer (Beyotime, Shanghai, China) and protease and phosphatase inhibitor cocktail (Thermo Fisher, USA) for 30 min. The protein concentration was assessed with a BCA assay kit (Beyotime, Shanghai, China). Protein extracts were separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (8–12% gels) and blotted onto PVDF membranes (Merck Millipore Ltd., Massachusetts, USA). After blocking with 5% fat-free milk, the membranes were incubated overnight at 4 °C with the following primary antibodies: HIF-1α (1:1000, ab179483), and Actin (1:1000, ab8226). The binding antibodies were detected by horseradish peroxidase-conjugated IgG (Multi Sciences) and visualized with enhanced chemiluminescence detection reagents (FDbio Science Biotech Co., Ltd., Hangzhou, China).
6
Western Blot Analysis of Angiomotin Protein
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Total proteins were extracted using sodium dodecyl sulfate lysis buffer (Beyotime, Jiangsu, People’s Republic of China), and bicinchoninic acid protein assay kit (Pierce, Rockford, lL, USA) was used to determine the concentration of total proteins. Equal amounts of total proteins were separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA) in 25 mM Tris base and 190 mM glycine at 50 V for 3 hours at 4°C. The membranes were subjected to 2 hours blocking in Tris-buffered saline containing 5% nonfat dried milk powder at 37°C and incubated with primary antibody (angiomotin, 1:8,000, Abcam, Cambridge, UK; GAPDH, 1:2,000, Abcam) for 1 hour at 37°C. The membranes were washed and subsequently treated with secondary antibody, goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1/4,000 dilution for 1 hour at room temperature and visualized by chemiluminescence.
7
EGFR and Integrin β1 Protein Detection
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Cells
separated at different time points were suspended in PBS by scraping
and were lysed by sodium dodecyl sulfate lysis buffer (Beyotime Biotechnology,
China). The protein concentration was determined using the bicinchoninic
acid protein assay kit (Beyotime Biotechnology, China). The separated
proteins were electroblotted onto nitrocellulose transfer membranes
(0.2 μm, Whatman, U.K.). The membranes were then blocked with
5% BSA in TBST (Tris-buffered saline and Tween 20) for 1 h and incubated
with primary antibodies (mouse anti-EGFR antibody, 1:100 dilution,
and mouse anti-integrin β 1 antibody, 1:200 dilution, Abcam)
at 4 °C overnight. The membranes were rinsed three times with
TBST for 10 min and then incubated with secondary antibody (HRP-labeled
goat anti-mouse IgG, dilution 1:2000, Beyotime Biotechnology). After
washing three times with the TBST for 10 min, protein bands were visualized
with the BeyoECL Plus kit (Beyotime Biotechnology). GAPDH was used
as a loading control.
separated at different time points were suspended in PBS by scraping
and were lysed by sodium dodecyl sulfate lysis buffer (Beyotime Biotechnology,
China). The protein concentration was determined using the bicinchoninic
acid protein assay kit (Beyotime Biotechnology, China). The separated
proteins were electroblotted onto nitrocellulose transfer membranes
(0.2 μm, Whatman, U.K.). The membranes were then blocked with
5% BSA in TBST (Tris-buffered saline and Tween 20) for 1 h and incubated
with primary antibodies (mouse anti-EGFR antibody, 1:100 dilution,
and mouse anti-integrin β 1 antibody, 1:200 dilution, Abcam)
at 4 °C overnight. The membranes were rinsed three times with
TBST for 10 min and then incubated with secondary antibody (HRP-labeled
goat anti-mouse IgG, dilution 1:2000, Beyotime Biotechnology). After
washing three times with the TBST for 10 min, protein bands were visualized
with the BeyoECL Plus kit (Beyotime Biotechnology). GAPDH was used
as a loading control.
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