Cells were lysed in lysis buffer (Beyotime, China) mixed with 1% protease inhibitors (Beyotime, China) to extract total protein. Mitochondrial fractions were extracted using a Cell Mitochondria Isolation Kit (Beyotime, China). The cell medium was centrifuged (800 × g, 5 min), and the supernatant was filtered for the detection of high mobility group box 1 protein (HMGB1), as described previously [17 (link)]. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Total cell lysates were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Amersham Biosciences, USA). Blots were probed with the primary antibodies against phosphorylated PKR-like ER kinase (p-PERK, 1 : 1000; CST, USA), GRP78 (1 : 1000; Abcam, UK), CHOP (1 : 1000; Abcam, UK), HMGB1 (1 : 10000; Abcam, UK), PARP (1 : 500; Abcam, UK), VDAC1 (1 : 1000; Abcam, UK), or GAPDH (1 : 1000; Tianjin Sun gene Biotech Co, China) and incubated (with rocking) at 4°C overnight. Membranes were probed with secondary antibodies and visualized using an enhanced chemiluminescence reagent (ECL, Amersham Biosciences, Piscataway, NJ, USA).
Gapdh
Manufactured by Sungene Biotech
Sourced in China, United Kingdom
GAPDH is a fundamental enzyme involved in the glycolytic pathway, responsible for the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. It is a widely used reference gene and protein expression marker due to its stable and ubiquitous expression across various cell types and experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Sungene Biotech (3 mentions)
Bcl2 associated x (bax), by Boster Bio (2 mentions)
Caspase 3, by Bioss Antibodies (2 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Vdac1, by Abcam (1 mentions)
Hmgb1, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Cell mitochondria isolation kit, by Beyotime (1 mentions)
Grp78, by Abcam (1 mentions)
Jagged 1, by Cell Signaling Technology (1 mentions)
Cd133, by Abcam (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
20 protocols using gapdh
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting Antibodies for EMT and Stemness
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The following antibodies were used for immunoblotting: the antibodies against E-cadherin (Cell Signaling), vimentin (Cell signaling), ZO1 (Cell signaling), Claudin-1 (Cell Signaling), β-catenin (Cell Signaling), ZEB1 (Cell Signaling), Slug (Cell Signaling), Snail (Cell Signaling), p-STAT3 (Cell Signaling), STAT3 (Cell Signaling), p-AKT (Cell signaling), AKT (Cell signaling), p-ERK (Santa Cruz), ERK (Santa Cruz), CD133 (Abcam), OCT4 (Cell signaling), Jagged-1 (Cell signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Sungene Biotech).
3
Western Blot Analysis of Signaling Proteins
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Cells were lysed by RIPA buffer supplemented with cocktail proteinase inhibitor. Then the samples were separated by SDS-PAGE gel electrophoresis, and electroblotted onto polyvinylidene fluoride membrane. After blocked with 5% BSA or nonfat milk, membranes were incubated overnight at 4°C with primary antibodies as indicated. Blots were developed with secondary horseradish peroxidase (HRP)-conjugated antibody, and signals were exposed onto x-ray films. The following antibodies were for Western blot: eIF3i(Abcam), phospho-AKT (p-AKT, Ser473; Cell Signaling technology), AKT (Cell signaling technology), phospho-ERK1/2 (p-ERK1/2, Thr202/Tyr204; Cell signaling technology), ERK1/2 (Cell signaling technology), VEGFR2 (Abcam), GAPDH (Tianjin sungene Biotech), Tubulin (Tianjin sungene Biotech).
4
Photodynamic Therapy Efficacy Analysis
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Pyropheophorbide α methyl ester (MPPa, C34H36N4O3) was obtained from Sigma-Aldrich (St. Louis, MO). The laser (630 nm) was purchased from Chongqing Jingyu Laser Technology Co., Ltd. (Chongqing, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone (Logan, UT). The Cell Counting Kit-8 (CCK-8) was procured from Dojindo Molecular Technologies (Kumamoto, Japan). Akt (catalogue number: 4691, dilution: 1:1000), phospho-Akt (catalogue number: 4060, Ser473, dilution: 1:2000), phospho-NF-κB p65 (catalogue number: 3033, Ser536, dilution: 1:1000) and NF-κB p65 (catalogue number: 8242, dilution: 1:1000) were obtained from Cell Signaling Technology (Danvers, MA). GAPDH was purchased from Sungene Biotech (Tianjin, China). Loading control was obtained from Beyotime (Shanghai, China). Trypsin and Actin-tracker Green were procured from Beyotime (Shanghai, China).
5
Western Blot Analysis of AKT and ELMO1
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Proteins extracted from cells were separated via SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was blocked with 5% skim milk and probed with antibodies against p-AKT-T308, p-AKT-S473, AKT (2965S, 4060S, and 9272S; Cell Signaling Technology, USA), ELMO1 (ab174298, Abcam, Cambridge, UK), and GAPDH (C1219, Tianjin Sungene Biotech, Tianjin, China) for the indicated proteins.
6
Western Blot Analysis of Cardiac Proteins
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Protein samples were prepared by homogenizing cardiac tissue in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors (Complete and PhosSTOP; Roche, Basel, Switzerland). Protein extracts from myocardial tissue were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% nonfat dry milk for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies, including Nr4a1 recombinant rabbit monoclonal antibody (Cat#MA5-32647, Thermo Fisher Scientific, 1:1000) and HK2 recombinant rabbit monoclonal antibody (Cat#ab209847, Abcam, 1:1000;). The housekeeping protein GAPDH (Cat#KM9002T, Sungene Biotech, 1:4000) was used as reference protein to normalize the target proteins during western blot analysis. After washing, the membranes were incubated with secondary antibodies antibody IgG (Cat# A0208, Beyotime Biotech, 1:1000) at the appropriate dilutions for 1.5 h at room temperature, and detected using the enhanced chemiluminescence substrate kit. The density of each band was quantified using Quantity One. Three biological repeats were performed for each sample.
7
Protein Extraction and Immunoblotting for STAT3 Signaling
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Protein extraction and SDS-PAGE were performed as described previously (14 (link)). Rabbit polyclonal antibody, β-actin and GAPDH were purchased from SunGene Biotech (SunGene, China). LepA-specific rabbit polyclonal antibody was obtained from HuaAn Biotech (HuaAn, China). Signal transducers and activators of transcription 3 (STAT3) and phosphorylation of STAT3 (p-STAT3) rabbit polyclonal antibodies were purchased from Elabscience Biotech (Elabscience, China).
8
Western Blot Analysis of EMT Markers
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Harvested cells were washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl/pH 7.4; 1% NP-40; 150 mM NaCl; 1 mM EDTA; 1 mM proteinase inhibitor; 1 mM Na3VO4; 1 mM NaF; 1 mM okadaic acid; and 1 mg ml−1 aprotinin, leupeptin, and pepstatin). Samples (30 μg protein) were separated on 8% SDS–PAGE gel and transferred to PVDF membranes at 4 °C (250 mA, 2 h). Membranes were blocked in 5% fat-free milk in TBST for 1 h at room temperature, and incubated with appropriate diluted primary antibodies GAPDH (Sungene Biotech, Tianjin, China, KM9002, 1 : 5000), Sox5 (Bioss Biosynth, bs-17136R, 1 : 1000), N-cadherin (Abcam, Catalogue# 124397, 1 : 1000), Twist1 (Abcam, ab50887, 1 : 50), Histone 3 (Abcam, ab8580, 1 : 1000), and E-cadherin (BD biosciences, Catalogue# 610181, 1 : 1000) were used for staining overnight at 4 °C, then washed 10 min three times, and incubated with HRP-conjugated anti-rabbit or anti-mouse antibody for 1 h at room temperature, washed 10 min three times. The blots were developed in ECL mixture and visualised by Imager.
9
Quantification of Osteoclast Proteins
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Proteins were extracted by lysing the osteoclasts with RIPA buffer (Aksomics, Shanghai) and quantified by a bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai). Protein extracts (equal quantities) were separated using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were then transferred onto a PVDF membrane (Whatman, UK). PVDF membranes were blocked with no fat milk and incubated at 4 °C overnight with primary antibodies against GAPDH (Tianjin Sungene Biotech, China), Ephrin B2 (Abcam Inc., USA) and SPHK1 (Cell Signaling Technology Inc., USA) at a dilution of 1:1000. After washing 3 times, the PVDF membranes were incubated with HRP-conjugated secondary antibodies (1:3000; Santa Cruz Biotechnology, USA) . Protein bands were visualized using ECL (Millipore, USA)and the densitometry readings were quantified using Image J software (NIH, USA).
10
Immunoblotting Analysis of Smac Protein
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Cytosol fractions were collected as described above for cytosolic cytochrome C
measurement. Smac expression was measured by immunoblotting. An equal amount of
protein solution from various treatments was loaded and analyzed by 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis,
proteins were transferred onto polyvinylidene fluoride membranes and blotted with
primary antibodies against smac (Abcam, UK) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; Tianjin Sungene Biotech Co., Ltd., China). Membranes were then
incubated with horseradish peroxidase-tagged secondary antibody (Earth, USA), and
signals were developed by an enhanced chemiluminescence reagent.
measurement. Smac expression was measured by immunoblotting. An equal amount of
protein solution from various treatments was loaded and analyzed by 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis,
proteins were transferred onto polyvinylidene fluoride membranes and blotted with
primary antibodies against smac (Abcam, UK) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; Tianjin Sungene Biotech Co., Ltd., China). Membranes were then
incubated with horseradish peroxidase-tagged secondary antibody (Earth, USA), and
signals were developed by an enhanced chemiluminescence reagent.
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