Total proteins from cell lines and tissues were extracted with RIPA buffer and then quanti ed by BCA analysis. Subsequently, 20 µg of total protein per sample (10 µL per lane) was separated using sodium lauryl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide gel) before the proteins were transferred to a PVDF membrane. After incubation with primary antibodies overnight, the membranes were then incubated with secondary antibody. Finally, target protein bands were detected using a chemiluminescence system. The antibodies used targeted the following proteins: AKT (Cell Signaling #9272s), Caspase 9 (Proteintech 10380-1-AP), Caspase 3 (Proteintech 66470-2-Ig), Caspase 8 (Proteintech 66093-1-Ig), p-ATK (Cell Signaling #4060), BAX (Proteintech 60267-1-Ig), Vimentin (Proteintech 10366-1-AP), N-cadherin (Proteintech 22018-1-AP), E-cadherin (Proteintech 20874-1-AP), GAPDH (Signalway Antibody #21612), and METTL3 (ABclonal A8370).
Gapdh
Manufactured by Signalway Antibody
Sourced in United States
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a key enzyme involved in the glycolytic pathway, catalyzing the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. As a core component of cellular metabolism, GAPDH plays a crucial role in energy production and is commonly used as a housekeeping gene or reference control in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 9, by Proteintech (3 mentions)
P atk, by Cell Signaling Technology (3 mentions)
E cadherin, by Proteintech (3 mentions)
N cadherin, by Proteintech (3 mentions)
Vimentin, by Proteintech (3 mentions)
Caspase 3, by Proteintech (3 mentions)
Bcl2 associated x (bax), by Proteintech (3 mentions)
Caspase 8, by Proteintech (3 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
Citrullinated histone h3 cith3, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Sds page protein loading buffer 5, by Beyotime (1 mentions)
Cleaved caspase 3, by Signalway Antibody (1 mentions)
Beas 2b, by Keygen Biotech (1 mentions)
Vimentin, by Signalway Antibody (1 mentions)
E cadherin, by Signalway Antibody (1 mentions)
N cadherin, by Signalway Antibody (1 mentions)
Penicillin, by Wisent (1 mentions)
Streptomycin, by Wisent (1 mentions)
8 protocols using gapdh
1
Quantitative Protein Expression Analysis
2
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detecting target protein levels, the cellular total proteins were resolved on an SDS denaturing polyacrylamide gel and then transferred onto a nitrocellulose membrane. Antibodies to CD151 (Santa Cruz, Dallas, TX, USA, Cat# sc-271216), PIK3C2A (Santa Cruz, Cat# sc-365290) or an endogenous control GAPDH (Signalway Antibody, College Park, MD, USA) were incubated with the membranes overnight at 4°C. The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Goat anti-mouse IgG, Genomapping, Tianjin, China). Protein expression was assessed by enhanced chemiluminescence and the bands were captured by a FluorChem FC2 Imaging System (Alpha Innotech, Kasendorf, Germany). The band intensity was analyzed by an AlphaView SA V3.4.0 (ProteinSimple, San Jose, CA, USA).
3
Quantifying Renal Transport Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, the tissue proteins were obtained and quantified after homogenizing in RIPA buffer containing protease and phosphatase inhibitor (NCM Biotech, Suzhou, China). An amount of 40 μg of protein lysate was resolved by SDS–PAGE gels, and the protein was then transferred onto PVDF membranes. The membranes were blocked in 5% skim milk, followed by incubation with the primary antibodies URAT1 (1:1000; Absin; Shanghai, China), GLUT9 (1:2000; LSBio, Seattle, WA, USA), or GAPDH (1:5000; Signalway Antibody, Greenbelt, MD, USA) overnight at 4 °C. Finally, the protein bands were visualized using a multifunctional imaging analysis system (FluorChem R ProteinSimple, Minneapolis, MN, USA) and quantified with intensities by ImageJ software.
4
Western Blotting Antibody Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was carried out as previously described [17 (link)], with the following antibodies: Mcl-1 (1:1000; Genetex, USA), cleaved-caspase 3 (1:1000; Cell Signaling Technology, USA), p-GSK-3β (Ser9) (1:1000; Cell Signaling Technology, USA), citrullinated histone H3 (Cit-H3, 1:1000; Abcam, USA), and GAPDH (1:5000; Signalway Antibody, Maryland, USA). ImageJ was used to calculate the grayscale values.
5
Western Blotting of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as described in a previous study with some modifications [20 (link)]. Protein extracts from cells or hippocampi were prepared in a modified RIPA buffer supplemented with protease inhibitors (200612, Signalway Antibody, Greenbelt, MD, USA). The BCA method was used to determine the concentration of protein. The protein extracts were boiled after being diluted in SDS-PAGE protein loading buffer (5×) (Beyotime, Shanghai, China) at a ratio of 4:1. Following separation, the proteins were transferred to polyvinylidene difluoride membranes and blocked in TBST buffer (20 mM Tris–HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween-20) with 5% non-fat milk at 37 °C for 1 h, and incubated at 4 °C with primary rabbit polyclonal antibodies (cleaved caspase-3, 49500, 1:500, Signalway Antibody; BCL-W, 40641, 1:1000, Signalway Antibody; SAPK/JNK (pThr183), 11249, 1:500, Signalway Antibody; SMAC/DIABLO, 39330, 1:500, Signalway Antibody; GAPDH, 21612, 1:3000, Signalway Antibody) overnight. After extensive rinsing, the membranes were incubated with the appropriate HRP-conjugated secondary antibody, then visualized using Super ECL Plus reagents. The gray values of the protein bands were quantified by the optical density using ImageJ software (1.41v, US National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from cell lines and tissues were extracted with RIPA buffer and then quantified by BCA analysis. Subsequently, 20 µg of total protein per sample (10 µL per lane) was separated using sodium lauryl sulphate–polyacrylamide gel electrophoresis (10% polyacrylamide gel) before the proteins were transferred to a PVDF membrane. After incubation with primary antibodies overnight, the membranes were then incubated with secondary antibody. Finally, target protein bands were detected using a chemiluminescence system. The antibodies used targeted the following proteins: AKT (Cell Signaling #9272s), Caspase-9 (Proteintech 10380-1-AP), Caspase-3 (Proteintech 66470-2-Ig), Caspase-8 (Proteintech 66093-1-Ig), p-ATK (Cell Signaling #4060), BAX (Proteintech 60267-1-Ig), Vimentin (Proteintech 10366-1-AP), N-cadherin (Proteintech 22018-1-AP), E-cadherin (Proteintech 20874-1-AP), GAPDH (Signalway Antibody #21612) and METTL3 (ABclonal A8370).
7
Neferine modulates NSCLC cell lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The NSCLC cells lines (A549, and H1299) and human normal lung epithelial cells BEAS-2B purchased from KeyGen BioTECH (Nanjing, China) were incubated in RPMI-1640 medium (KeyGEN BioTECH, NanJing, China) contains 10% fetal bovine serum (FBS; WISENT corporation, Wisent, Canada), as well as 1% penicillin (WISENT) and 1% streptomycin (WISENT). The cells were incubated in Heracell 150i Incubator (Thermo Fisher Scienti c, Waltham, MA, USA) at 37°C in a 5% CO 2 atmosphere. Trypin was purchased from WISENT. Neferine (Nef, C 38 H 44 N 2 O 6 , Molecular weight: 624.8, Purity> 95%), obtained from Nanjing University of Chinese Medicine (Nanjing, China), and the molecular structure were shown in Figure 1A. Nef were dissolved in dimethyl sulfoxide (DMSO) to produce 10 mM stock solution and stored at -80°C. The primary antibodies including B-cell lymphoma-2 (BCL2) (#48496), BCL2 associated X protein (BAX) (#29057), ROCK1 (#48890), GAPDH (#21612), E-cadherin (#40860), N-cadherin (#48495), Vimentin (#41531) were purchased from Signalway Antibody (MD, USA), and the HRP-conjugated Goat anti-Rabbit (#S0001) were purchased from A nity Biosciences (Cincinnati, OH, USA). All antibodies were used in accordance with the instructions. Recombinant human transforming growth factor-β1 (TGF-β1) was purchased from STEMCELL Technologies (Vancouver, BC, Canada).
8
Quantitative Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from cell lines and tissues were extracted with RIPA buffer and then quanti ed by BCA analysis. Subsequently, 20 µg of total protein per sample (10 µL per lane) was separated using sodium lauryl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide gel) before the proteins were transferred to a PVDF membrane. After incubation with primary antibodies overnight, the membranes were then incubated with secondary antibody. Finally, target protein bands were detected using a chemiluminescence system. The antibodies used targeted the following proteins: AKT (Cell Signaling #9272s), Caspase 9 (Proteintech 10380-1-AP), Caspase 3 (Proteintech 66470-2-Ig), Caspase 8 (Proteintech 66093-1-Ig), p-ATK (Cell Signaling #4060), BAX (Proteintech 60267-1-Ig), Vimentin (Proteintech 10366-1-AP), N-cadherin (Proteintech 22018-1-AP), E-cadherin (Proteintech 20874-1-AP), GAPDH (Signalway Antibody #21612), and METTL3 (ABclonal A8370).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!