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).
Anti pkm
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-PKM is a primary antibody that recognizes the pyruvate kinase M1/M2 (PKM) enzyme, which plays a key role in glycolysis. It can be used to detect and quantify PKM expression in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Pi 31460, by Thermo Fisher Scientific (2 mentions)
Anti pkm2, by Cell Signaling Technology (2 mentions)
Anti cox 4, by Proteintech (2 mentions)
Anti calreticulin, by Cell Signaling Technology (2 mentions)
Anti pkm1, by Cell Signaling Technology (2 mentions)
Anti p70 s6 kinase, by Cell Signaling Technology (2 mentions)
Anti human specific ldha, by Cell Signaling Technology (2 mentions)
Anti ldhb, by Abcam (2 mentions)
Anti human and rabbit ldha, by Abcam (2 mentions)
Anti golgin, by Cell Signaling Technology (2 mentions)
Pi31430, by Thermo Fisher Scientific (2 mentions)
Anti gpt2, by Santa Cruz Biotechnology (2 mentions)
Anti phgdh, by Merck Group (2 mentions)
Anti pc, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Sodium dodecyl sulfate lysis buffer, by Beyotime (1 mentions)
Non fat powdered milk, by Sangon (1 mentions)
6 protocols using anti pkm
1
Protein Expression Analysis by Western Blotting
2
Western Blot Analysis of Metabolic Enzymes
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Cell lysates were prepared and subjected to WB analysis as performed previously51 (link),52 (link). The following commercial antibodies were used: anti-β-actin (Sigma A1978), anti-PKM2 (Cell Signaling Technology 3198S), anti-PKM1 (Cell Signaling Technology 7067), anti-PKM (Abcam AB118499), anti-human specific LDHA (Cell Signaling Technology 2012S), anti-LDHB (Abcam AB75167), anti-human and rabbit LDHA (Abcam AB135396), anti-p70 S6 kinase (Cell Signaling Technology 2708T), anti-calreticulin (Cell Signaling Technology 12238T), anti-golgin (Cell Signaling Technology 13192), anti-COX IV (Proteintech 11242-1-AP), anti-GPT2 (Santa Cruz sc-398383), anti-PHGDH (Sigma HPA021241), and anti-PC (Santa Cruz sc-271493), horseradish peroxidase-conjugated secondary antibodies anti-mouse (Fisher PI31430) and anti-rabbit (Fisher PI31460). All the primary antibodies were used at a 1:500 dilution in 5% non-fat milk in TBST. Secondary antibodies were used at a 1:3000 dilution in 5% non-fat milk in TBST.
3
Western Blot Analysis of Metabolic Enzymes
4
m6A Methylation Protein Binding Assay
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RIP assay was conducted by using Z-Magna RIPTM RNA-binding Protein Immunoprecipitation kit (Millipore Corporation, USA). Cells lysates were obtained by treated cells with lysis buffer, Next, cell lysates were immunoprecipitated with anti-m6A (1mg/ml; Abcam, CA, USA), anti-METTL3 (1mg/ml; Abcam), anti-ENO1 (850μg/ml; Abcam), anti-PKM (550μg/ml; Abcam), anti-IGF2BP3 and negative control anti-IgG (1mg/ml; Abcam). Finally, the RNA complexes were extracted for RT-qPCR. The experiment was performed in triplicate.
5
Protein Extraction and Western Blot Analysis
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The cell protein was extracted using RIPA buffer (Thermo). Protein concentration was determined by BCA Protein Assay (Beyotime). Total protein was loaded, fractionated by SDS-PAGE, transferred to PVDF membrane, and probed with anti-β actin (Abcam), anti-PKM (Abcam), anti-PFKM (Cell Signaling Technology), anti-PDHB (Abcam), and anti-IDH2 (Cell Signaling Technology). Signal was detected using Chemiluminescence imaging system (Tanon, Shanghai, China).
6
Western Blot Analysis of hADSC Treated with Linoleic Acid
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Briefly, hADSC were treated with 25 μmol LA or Ctrl for 24 h and 48 h (or 80 h) then lysed using RIPA buffer containing protease and phosphatase inhibitors at 4° C, followed by centrifugation for 10 min. The supernatant was collected and sample buffer (5×) was added at a ratio of 5:1. Samples were mixed well and boiled for 10 min, followed by storage at −40° C. Proteins were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes that were blocked by incubation with 2.5% dry skim milk, followed by overnight incubation with primary antibodies diluted in 2.5% dry skim milk at 4° C.
The following antibodies were used: monoclonal rabbit anti-RUNX2, anti-LPL, anti-P16ink4a, anti-GAPDH, anti-MMP14, anti-PKM, anti-PFKP, anti-AMPK, anti-p- AMPK (Abcam) at 1:1000 dilutions. The blots were then incubated with the secondary mouse or rabbit antibodies at room temperature for 1 h. Proteins were detected using the BioSpectrum 600 system. The western blots repeated 3 independent experiments.
The following antibodies were used: monoclonal rabbit anti-RUNX2, anti-LPL, anti-P16ink4a, anti-GAPDH, anti-MMP14, anti-PKM, anti-PFKP, anti-AMPK, anti-p- AMPK (Abcam) at 1:1000 dilutions. The blots were then incubated with the secondary mouse or rabbit antibodies at room temperature for 1 h. Proteins were detected using the BioSpectrum 600 system. The western blots repeated 3 independent experiments.
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