Harvested cells were lysed by RIPA buffer. After sonication, samples were centrifuged at 12 000 g for 15 min at 4°C. Total protein concentration was determined by applying DC Protein Assay Kit I (Bio‐Rad, USA). Proteins were transferred to Hybond nitrocellulose membranes (USA) after separation on 12% SDS‐PAGE. 5% skim milk was added to seal the membrane in Tris‐buffered saline (pH 7.5) at room temperature. The membrane was incubated with the primary antibody overnight at 4°C, followed by 4‐h incubation with the secondary antibody at room temperature. Protein bands were developed by ECL kit (Millipore, USA). Protein levels were assessed by ImageJ (USA). Primary antibodies including anti‐CD133, anti‐CD44, anti‐Oct‐4, anti‐HK2, anti‐PKM2, anti‐LDHA, anti‐β‐actin, and secondary antibody anti‐IgG were purchased from Abcam (UK).16
Anti pkm2
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-PKM2 is a protein-specific antibody that recognizes Pyruvate Kinase M2 (PKM2), an enzyme involved in glycolysis. This antibody can be used for the detection and analysis of PKM2 in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti hk2, by Abcam (4 mentions)
Anti ldha, by Abcam (4 mentions)
Anti glut1, by Abcam (3 mentions)
Anti β actin, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Ip cell lysis buffer, by Beyotime (2 mentions)
Anti ha, by Abmart (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Irdye 680rd, by LI COR (2 mentions)
Anti cd133, by Abcam (1 mentions)
Anti oct4, by Abcam (1 mentions)
Anti cd44, by Abcam (1 mentions)
Dc protein assay kit 1, by Bio-Rad (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Fitc anti mouse b220, by BioLegend (1 mentions)
Anti p erk, by Abcam (1 mentions)
Hrp labeled goat anti mouse igg, by Beyotime (1 mentions)
Anti erk, by Abcam (1 mentions)
14 protocols using anti pkm2
1
Western Blot Analysis of Stem Cell Markers
2
Analyzing Protein Expression in PI3K/AKT Pathway
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Equal amounts of extracted proteins were injected into sodium dodecyl sulfate polyacrylamide gels and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010, United States). The membranes were then treated by washing with TBST buffer, followed by blocking with 5% skim milk powder and incubation with primary antibody. After 1 d, the membranes were incubated with a suitable secondary antibody at 37 °C for 30 min, followed by three washes with TBST for 5 min each time. Finally, ECL luminescence reagent (Novozymes, E412-01, China) was dropwise added to the front of the PVDF membrane and the developed films were fixed using an automatic film washer in a darkroom. The resulting films were scanned and archived. The antibodies used in this experiment were anti-PKM1 (Abcam, Cambridge, MA, United States), anti-PKM2 (Abcam, Cambridge, MA, United States), anti-AKT (Proteintech, 60203-2-Ig, China), anti-p-AKT (Proteintech, 28731-1-AP, China), and anti-GAPDH (Proteintech, 60004-1-Ig, China). The PI3K/AKT pathway inhibitor used was Ly294002 (MCE, HY-10108, China).
3
Western Blot Analysis of Glycolytic Enzymes
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The method as followed the previous article.19 Cells were gathered and treated with lysis buffer. Equal amounts of total proteins (20 μg) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (EMD Millipore). The membrane was blocked with 5% skimmed milk and incubated with the corresponding primary antibodies overnight at 4°C. After washing with 1× TBST three times, the membrane was incubated with the horseradish peroxidase‐conjugated secondary antibody (goat anti‐rabbit IgG) for 1 h at room temperature. Following three washes with 1× TBST, the immunoreactivity was detected using an enhanced chemiluminescence method and captured using a chemiluminescence imaging system. The primary antibodies used were Anti‐HK‐2, Anti‐PKM2, Anti‐LDHA, and Anti‐β‐Actin (all rabbit anti‐human), and the secondary antibody was goat anti‐rabbit IgG. All of the antibodies were purchased from Abcam.
4
Optimized Antibody Panel for Immunoblotting and Flow Cytometry
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The following antibodies were used for immunoblotting: Cell Signaling Technology, anti-p38 (Cat#: 8690), anti-p-p38 (Cat#: 4511), anti-p65 (Cat#: 8242), anti-p-p65 (Cat#: 3033), anti-JNK (Cat#: 9252), anti-p-JNK (Cat#: 4668), anti-Erk (Cat#: 4695), anti-p-Erk (Cat#: 4370), anti-PKM2 (Cat#: 4053), anti-STAT3 (Cat#: 12640); Abcam, anti-Pyk2 (Cat#: 228477); Santa Cruz Biotechnology, anti-p-Pyk2 (Cat#: sc-81512); Beyotime Institute of Biotechnology, anti-β-actin (Cat#: AA128), HRP labeled Goat Anti-Rabbit IgG (Cat#: A0208), HRP-labeled Goat Anti-Mouse IgG (Cat#: A0216). The following antibodies purchased from Biolegend were used for flow cytometry: FITC anti-mouse B220 (Cat#: 103206), BV421 anti-mouse CD11c (Cat#: 117330), FITC anti-mouse F4/80 (Cat#: 123108), APC anti-mouse CD86 (Cat#: 105012), PE anti-mouse CD40 (Cat#: 124610), PE anti-mouse GL7 (Cat#: 144607), APC anti-mouse CD95 (Cat#: 152604), APC anti-mouse CXCR5 (Cat#: 145506), PE anti-mouse PD-1 (Cat#: 135206), APC/Cy7 anti-human CD19 (Cat#: 302218), FITC anti-human CD14 (Cat#: 367116), BV421 anti-human CD11c (Cat#: 301628), APC anti-human CD86 (Cat#: 374208), PE anti-human CD40 (Cat#: 334308). All the antibodies for flow cytometry were used at a 1:100 dilution.
5
Western Blot Analysis of Metabolic Regulators
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Cells were washed twice with cold phosphate buffer saline (PBS) and lysed using cell lysis buffer. Protein concentration was measured with a BCA assay kit (Beyotime). Protein extracts were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidenefluoride membranes. Membranes were incubated with specific primary antibodies overnight at 4°C after blocking in 5% non-fat milk as follows: anti-c-Myc, anti-HIF-1α, anti-HK2, anti-Bcl-2, anti-LDHA, anti-phosphorylated STAT3 (Tyr705) and anti-STAT3 (1:1000, CST); anti-Glut1, anti-Glut3, anti-PFK, and anti-PKM2 (1:1000, Abcam); anti-β-actin (1:4000, Sigma). The membranes were incubated with horseradish peroxidase conjugated secondary antibody for 1 h at room temperature, and visualized with enhanced chemiluminescence. The aimed protein levels were normalized to the level of β-actin.
6
Detecting Metabolic Regulators via Western Blot
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Whole‐cell extracts were obtained using RIPA buffer composed of 50 mM Tris–HCl (pH 7.5), 15 mM NaCl, 1% NP‐40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitors. In the Western blotting analysis, 30 μg of total protein was loaded onto a 12.5% sodium dodecyl sulfate‐polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked at 4°C overnight in TBS containing 5% Phospho Blocker Blocking Reagent and 0.2% Tween‐20 and then incubated at 4°C overnight with the following primary antibodies (Abs): anti‐IDH2 (1:500 dilution), anti‐Hif‐1α (1:200 dilution, Santa Cruz Biotechnologies, Dallas, TX, USA), anti‐PHD2 (1:500 dilution, Invitrogen, Waltham, MA, USA), anti‐GLUT1 (1:200 dilution), anti‐TIGAR (1:1,000 dilution), anti‐anti‐TKT (1:2,500 dilution), anti‐CTPS1 (1:1,000 dilution), anti‐PKM2 (1:1,000 dilution), anti‐LDHA (1:2,000 dilution), anti‐IDH1 (1:500 dilution; Abcam, Tokyo, Japan), anti‐ME1 (1:500 dilution), and anti‐G6PD (1:100 dilution). The blots were incubated with a peroxidase‐labeled secondary Ab for 1 h. After PBS washing, the signals were detected using enhanced chemiluminescence reagents with the ECL plus Western Blotting Detection System and analyzed using the LAS 3000 system (GE Healthcare, Los Angeles, CA, USA).
7
Quantification of Apoptosis-Related Proteins in Mouse Tumors
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Samples of mouse tumor tissue were ground at low temperature, and the tissue seriflux and cells in each group were added to RIPA lysate buffer. After incubation for 40 min on ice, the solution was centrifuged (13400 g for 15 min), and the supernatant was collected. The extracted proteins were quantified using the BCA method (Beyotime, China). Next, a 40 μg aliquot of total protein from each sample was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Roche, Basel, Switzerland). After blocking, the membranes were incubated with primary antibodies (1: 1000) against the target proteins overnight at 4°C and then subsequently incubated with a secondary antibody (1: 2000) for 2 h. The immunostained protein bands were detected by treatment with ECL solution (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies used (anti-caspase 3, anti-HK2, anti-PKM2, and anti-LDHA) and secondary antibody (anti-GAPDH) were purchased from Abcam (Cambridge, MA, USA).
8
Immunofluorescence Analysis of Spinal Cord Tissue
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For immunofluorescence analysis, spinal cord cryosections were permeabilized with 0.2% Triton X-100 in PBS for 20 min, blocked with 2% BSA in PBS for 30 min, and then incubated overnight at 4°C with primary antibodies. Subsequently, sections were incubated for 2 h at room temperature with species-specific Alexa Fluor–conjugated secondary antibodies (Abcam). CNS tissue sections were incubated for 1 h with the fluorescent myelin stain FluoroMyelin Green (1:200; Invitrogen). Slides were rinsed in PBS and coverslipped in ProLong Gold antifade reagent with DAPI (Invitrogen). For T cell immunofluorescence, cells were incubated on poly-L-lysine–coated coverslips, fixed (4% PFA), and permeabilized. After incubation with primary and secondary antibodies, coverslips were washed and mounted onto microscope slides using a DAPI-containing mounting medium. The following primary antibodies were used: anti-PKM2 (1:200; Abcam) and anti-STAT3 (1:200; Cell Signaling). The slides were visualized with a high-resolution SP5 confocal microscope (Leica) and image analysis performed on Fiji software.
9
Molecular Profiling of Metabolic Pathways
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IR780 was obtained from Sigma-Aldrich (Darmstadt, Germany), 2DG was obtained from MedChemExpress (MCE, Monmouth Junction, USA), human recombinant transforming growth factor-β1 (anti-TGF-β1) antibody was obtained from Peprotech (Cranbury, USA) and sulfobromophthalein disodium salt hydrate (anti-BSP) was from Sigma-Aldrich. Antibodies of hexokinase-II (anti-HK2), anti-pyruvate kinase isozyme M2 (anti-PKM2), anti-lactate dehydrogenase A (anti-LDHA), anti-glucose transporter-1 (anti-GLUT1), anti-collagen type I alpha1 (anti-COL1A1), anti-fibronectin, anti-alpha-smooth muscle actin (anti-α-SMA) and anti-solute carrier organic anion transporter family member 2A1 (anti-SLCO2A1) antibodies were purchased from Abcam (Cambridge, UK). Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) was obtained from Proteintech (Rosemont, USA). Goat anti-rabbit secondary antibodies were obtained from LI-COR Biosciences (Lincoln, USA).
10
Protein Quantification and Western Blot Analysis
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Relative protein were extracted from tissue samples or cultured cells, and protein concentration was quantified by Lowry’s protein assay (Bio-Rad, Hercules, CA, USA) [19 (link)]. All protein samples were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes.The primary antibodies used in this study were anti-BUB1B(#4116, Cell Signaling Technology, Beverly, MA, USA), anti-ZNF143(#PA5-72,658, Invitrogen, Waltham, MA, USA), anti-GLUT1(#ab115730, Abcam, Cambridge, UK), anti-LDHA(#ab101562, Abcam, Cambridge, UK), anti-PKM2 (#ab137852, Abcam, Cambridge, UK), anti-HK2 (#ab137852, Abcam, Cambridge, UK) and anti-Actin (#23,600-1-AP, Proteintech, China) antibodies. Western blot analysis was analyzed by Image J software.
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