The proteins of liver samples were prepared with radioimmunoprecipitation assay lysis buffer and phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was detected with a BCA Protein Assay kit (Beyotime Institute of Biotechnology) with a loading mass of 20 µg/well. The samples were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% nonfat milk in PBST buffer (PBS +0.1% Tween 20) for 2 h at room temperature, then incubated with primary antibodies overnight at 4°C and secondary antibodies (Beijing ZhongShan Golden Bridge Biotechnology Co., Ltd Beijing, China; 1:5,000) at 37°C for 0.5 h. The following primary antibodies were used: mTOR (1:1,000), beclin1 (1:1,000), MAP1LC3B (1:1,000), caspase-3 (1:1,000) and GAPDH (cat. no. SC-32233; 1:10,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Protein bands were visualized by Enhanced Chemiluminescence kit (Beyotime Institute of Biotechnology). Integral optical density (IOD) value measured with Image-Pro Plus (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the relative expression of the target protein (ratio) using the following equation: Ratio=IOD target protein/IOD GAPDH.
Gapdh
Manufactured by Media Cybernetics
Sourced in United States
GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) is an enzyme involved in the glycolytic pathway. It catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, a key step in the production of energy within cells.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus 6, by Media Cybernetics (4 mentions)
Image pro plus software, by Media Cybernetics (3 mentions)
Image pro plus, by Media Cybernetics (2 mentions)
Image pro plus software version 6, by Media Cybernetics (2 mentions)
Mmp 9, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Media Cybernetics (2 mentions)
Gapdh, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Gapdh, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Lysis buffer, by Genechem (1 mentions)
Bicinchonic acid assay, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by R&D Systems (1 mentions)
Sirt1, by Merck Group (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Phosphorylated mtor, by Cell Signaling Technology (1 mentions)
H4k16ac, by Merck Group (1 mentions)
13 protocols using gapdh
1
Western Blot Analysis of Liver Proteins
2
Evaluating Liver eNOS Expression
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The L02 cells or middle right liver tissues of rats were lysed in lysis buffer (Genechem Co., Ltd., Shanghai, China). Lysates were centrifuged at 7,500 × g for 10 min at 4°C, and the supernatant was collected. Total protein levels in supernatant samples were quantified using a bicinchonic acid assay (Thermo Fisher Scientific, Inc.). Samples (50 µg protein) underwent 10% SDS-PAGE. Proteins were then electroblotted onto a polyvinylidene difluoride membranes. The membrane was blocked with 5% fat-free milk at 4°C overnight, followed by incubation with rabbit eNOS primary antibody (1:1,000; cat. no. MAB9028; R&D Systems, Inc., Minneapolis, MN, USA) at 37°C for 2 h. Membranes were washed three times with TBS-T, then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. sc-2065; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The density of the corresponding bands was measured quantitatively using Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and corrected by reference to the value for GAPDH (1:1,000; cat. no. NB300-221; R&D Systems, Inc.).
3
Protein Expression Analysis by Western Blot
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Western blotting was performed as previously described (Zhou et al., 2011 (link)). The primary antibodies used were SIRT1 (07-131; EMD Millipore), SIRT3 (5490; Cell Signaling Technology), AMPKα (9957; Cell Signaling Technology), phosphorylated AMPKα (9957; Cell Signaling Technology), mTOR (2983; Cell Signaling Technology), phosphorylated mTOR (2971; Cell Signaling Technology), GAPDH (5174; Cell Signaling Technology), MMP2 (sc13595; Santa Cruz Biotechnology, Inc.), MMP9 (ab38898; Abcam), H3K9ac (ab10812; Abcam), H3 (ab1791; Abcam), H4K16ac (07-329; EMD Millipore), and H4 (05-858; EMD Millipore). Western blots were quantified densitometrically using Image Pro-Plus software (Media Cybernetics), the intensity values were calculated relative to GAPDH, H3, or H4, and the values were normalized to AL-Con values.
4
Hippocampal α5GABAA Expression Analysis
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After the tone-cued test was conducted, the mice were euthanized and their hippocampus tissue was isolated. Western blotting was performed as previously described [20 (link)]. The primary antibodies used were α5GABAA (ab10098; Abcam) and GAPDH (5174; Cell Signaling Technology). Western blots were quantified densitometrically using Image Pro-Plus software (Media Cybernetics), and the intensity values of α5GABAA were calculated relative to GAPDH.
5
Immunoblot Analysis of Lung Tissue
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Immunoblot analyses of lung tissue were performed on total lysates as described by Ding et al. (2019) (link) using phospho-signal transducer and activator of transcription (STAT) 3 (p-STAT3; catalog number, ab76315; Abcam, Cambridge, United Kingdom), STAT3 (ab68153; Abcam), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA; ab40776; Abcam), phospho-mitogen-activated protein kinase (MAPK) 1 (p-MAPK1; ab201015; Abcam), MAPK1 (ab184699; Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60004-1-Ig, Proteintech) as primary antibodies and then incubation with the corresponding secondary antibodies. Protein bands were detected by an electrochemiluminescence reagent (NCM Biotech, Beijing, China). The intensity of protein bands was analyzed using Image-Pro Plus (Media Cybernetics, Rockville, ML, United States) and presented as the ratio to GAPDH.
6
Western Blot Analysis of Oxidative Stress Markers
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The thawed samples were homogenized in total protein extraction buffer with added proteinase inhibitor. After determination of the protein concentration, denatured protein was subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). After blocking, the membranes were probed with rabbit polyclonal antibodies anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HO-1 and anti-Bcl-2 (Abcam, UK), anti-Bax (CST, Danvers, MA, USA), anti-GAPDH (Proteintech Group, Inc, Wuhan, China), and anti-β-actin (Guge, Wuhan, China) at 4 °C overnight, followed by horseradish-peroxidase-conjugated secondary anti-rabbit antibodies (Proteintech Group, Inc, China) at room temperature for 1 h. The specific bands were visualized with electrochemiluminescence (ECL) reagent and captured by G:BOX Chemi XT4 (Syngene, Frederick, MD, USA). For quantification, the integral optical density (IOD) values of Nrf2, HO-1, Bcl-2, Bax, GAPDH, and β-actin were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics Inc, Silver Spring, MD, USA).
7
Western Blot Analysis of EMT Markers
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Cells were lysed with RIPA buffer to extract proteins, which were separated with 12% SDS-PAGE, and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). The membrane was incubated with PBS containing 5% milk at room temperature for 3 h. The PVDF membrane was incubated with primary antibodies including SALL4, E-cadherin, N-cadherin, fibronectin, vimentin, MMP2, MMP9, and GAPDH (all from Abcam, Cambridge, MA, USA) at room temperature for 3 h, and then with secondary antibody (Abcam) at room temperature for 1 h. SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA) was then used to detect signals according to the manufacturer’s instructions. The relative protein expression was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA), represented as the density ratio versus GAPDH.
8
Analyzing Inflammatory Markers in Kidney Tissue Lysates
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Kidney tissues were lysed in RIPA lysis buffer (Pierce; USA) and total protein of the lysates was measured using a BCA assay kit (Nanjing Jiancheng Bioengineering Institute). Lysates were separated by 12% SDS-polyacrylamide gels and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked with 5% nonfat dried milk in TBST buffer for 1 h at room temperature. Blotted membranes were incubated with the following primary antibodies overnight at 4°C: anti-IL-6 (1:200; Invitrogen, USA), anti-TNF-α (1:200; Invitrogen), anti-NF-κB (1:200; Invitrogen), and GAPDH (1:200; Invitrogen). Then, the membranes were washed with TBST and incubated with HRP-labeled goat anti-mouse IgG antibody for 1 h at room temperature. Visualization was carried out with the Pierce ECL Western blotting substrate. The relative protein levels were assayed using Gel-Pro 4.0 software (Media Cybernetics, Inc., USA) and normalized to GAPDH.
9
Western Blot Analysis of Notch Pathway Proteins
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Total protein was extracted with Radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors, and the protein concentration was measured by the BCA assay. Equal quantities of the total protein were loaded into wells and separated using 10% SDS-PAGE, and then transferred electrophoretically to polyvinylidene fluoride membranes. After blocking in 5% nonfat dry milk for 2 h, the membranes were incubated overnight with primary antibodies at 4°C. The dilution ratios of primary antibodies in the experiment were as follows: DLL4 (1: 1000), Notch1 (1: 1000), Notch4 (1: 1000), and GAPDH (1: 1000) (lot 00093663, Proteintech Co., Ltd., United States). Next, the membranes were washed with PBST, then the secondary antibodies were added and incubated at 37°C for 1 h. The gray values of the bands were quantified and normalized to GAPDH by the Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.).
10
Real-time PCR and Western Blotting Analysis
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Real-time PCR was performed on an ABI StepOnePlus™ Real-Time PCR System (ABI, Carlsbad CA, USA). The primers used for amplification of chemotactic factors and TGF-β molecules were listed in Supplementary Table 5 . In western blotting assay, proteins were extracted from HASMCs or human thoracic aorta tissues and subjected to immunoblotting with rabbit polyclonal antibodies against p-ERK、p-JNK、p-GSK, mouse monoclonal antibodies against pSMAD2、pSMAD3 (1:1000, Santacruz, California, USA). GAPDH was used as internal control. Gel-Pro analyzer 4.0 software package was used to analysis the grey scales of target proteins, which were standardized against that of GAPDH (Media Cybernetics, Silver Spring, MD, USA).
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