Cultured HTB75 and OVCAR3 cell lines including parental, mock-transduced, and MALAT1-transduced cells were seeded and collected for Western blotting. Cell lysates were denatured and electrophoresed by 12% SDS-PAGE, as previously described. Primary antibodies against PI3K, phosphorylated (p)-PI3K, Akt, p-Akt, NFκB, p-NFκB, P38, p-P38, Bcl-2, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from ElabScience Biotechnology (Houston, TX, USA), and GAPDH/β-actin were used as a loading control. Appropriate secondary antibodies were used to detect proteins, as previously described. An ImageQuantTM LAS 4000 analyzer (GE Healthcare Life Science, Pittsburgh, PA, USA) was used to quantify protein expression levels.
Gapdh
Manufactured by Elabscience
Sourced in United States, China
GAPDH is an enzyme that catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate during the glycolysis process. It plays a crucial role in energy metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
β actin, by Elabscience (1 mentions)
Las 4000, by Cytiva (1 mentions)
P erk, by Proteintech (1 mentions)
P jnk, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β catenin, by Elabscience (1 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
C digit chemiluminescent western blot scanner, by LI COR (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Phosphorylated p65, by Cell Signaling Technology (1 mentions)
Hsp70, by Abcam (1 mentions)
Cd105, by Elabscience (1 mentions)
Wnt5a, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides lps escherichia coli o111 b4, by Merck Group (1 mentions)
Slc7a11, by Affinity Biosciences (1 mentions)
Bca protein detection kit, by Keygen Biotech (1 mentions)
Goat anti rabbit igg, by Elabscience (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
12 protocols using gapdh
1
Protein Expression Profiling of MALAT1-Transduced Cancer Cells
2
RIPA Lysis Protein Extraction Immunoblotting
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Using Radioimmunoprecipitation assay buffer (RIPA) lysate, mouse tissues were lysed for 30 min on ice, and protein supernatants were collected by centrifuging at 4 °C and 12,000 rpm for 15 min. The concentration of protein was determined using the BCA assay. The proteins were denatured by adding a loading buffer and heating the mixture for 10 min. The total amount of protein was limited to about 40 μg. After 30 min of electrophoresis at 80 V, the voltage was increased to 120 V for an additional 90 min, and then electroconversion was performed. BSA blocked the PVDF membrane for 2 hours at room temperature. The required antibodies (ERK, proteintech, Cat No. 11257-1-AP, 1:1000; p-ERK, proteintech, Cat No. 28733-1-AP, 1:2000; JNK, proteintech, Cat No. 24164-1-AP, 1:1000, p-JNK, proteintech, Cat No. 80024-1-RR, 1:1000; P38, proteintech, Cat No. 14064-1-AP, 1:1000; p-P38, proteintech, Cat No. 28796-1-AP, 1:1000 and GAPDH, Elabscience, Cat No. E-AB-20059, 1:5000) were diluted in the primary antibody’s dilution solution and incubated overnight at 4°C.After three TBST treatments, the secondary antibody was added and incubated for 2 h at room temperature. The ECL luminescence developer was configured according to specifications, exposed using a chemiluminescence instrument, and the results were analyzed.
3
Western Blot Analysis of Signaling Proteins
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After specific treatment incubation, the cells were collected and lysed in RIPA buffer with protease inhibitor cocktail (Roche). The total protein concentration was estimated by using Bradford protein estimation assay and an equal concentration (30 µg) was loaded in each well of 12% SDS-PAGE. After running, the gel was transferred onto the PVDF membrane (Merck Millipore) by using the Trans-Blot® Turbo™ Blotting System (Bio-Rad-Hercules, CA, USA). The membrane was then blocked with 5% Bovine serum albumin (BSA) in TBST for 1 hour at room temperature and then incubated with primary antibody overnight at 4℃. The membrane was washed with TBST 4–5 times and incubated with secondary antibody for 1 h at room temperature. Primary antibodies used in this study were CHK1, CHK2, Cyclin D1, p53, AKT, p38, MEK1, mTOR, p62, PI3K, LC3, CDK6, SAPK/JNK, STAT3, GSK3α, β-catenin, Beclin, and GAPDH (Elabsciences, Houston, TX, USA). The chemiluminescent signals were detected and processed in C-Digit Chemiluminescent Western Blot Scanner (LI-COR Lincoln, NE, USA).
4
Adipogenic Differentiation of Human Mesenchymal Stem Cells
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Lipopolysaccharides (LPS) (Escherichia coli O111: B4) were purchased from Sigma-Aldrich (United States). An adipogenic differentiation medium of human mesenchymal stem cells was obtained from Procell Life Science &Technology Co., Ltd., (Wuhan, China). Antibodies for flow cytometry—CD73, CD90, CD105, and CD45—were purchased from Elabscience (Wuhan, China). Antibodies for Western blot were procured from suppliers as follows: CD9, CD81, and GAPDH were acquired from Elabscience (Wuhan, China); ALIX and HSP70 were obtained from Abcam (Cambridge, United Kingdom); Wnt5a, p65, and phosphorylated-p65 were bought from Cell Signaling Technology (United States); anti-rabbit HRP-conjugated antibody was from Absin (China); PKH67 was from Fluorescence (China); and 4′,6-diamidino-2-phenylindole (DAPI) were from Solarbio (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were obtained from Absin (China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-10 were obtained from Elabscience (China).
5
Molecular Profiling of H9C2 Cells
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The H9C2 cells were collected in a 25 cm culture flask and cleaved to obtain total protein using a pre-cooled RIPA lysis buffer containing 1 mM PMSF. Total protein concentration was determined using a BCA protein detection kit (KeyGEN, Jiangsu, China). The same amount of protein was separated by 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was closed with rapid sealing solution at room temperature for 30 min and washed with PBST. TLR4 (1:1000; Santa Cruz, CA, USA), NF-κB (1:2000; Abbkine, Wuhan, China), SLC7A11 (1:500; Affinity, PA, USA), p53 (1:1000; Biolead, Beijing, China), GPX4 (1:1000; Abmart, Shanghai, China), and GAPDH (1:5000; Elabscience, Wuhan, China) were incubated at 4 °C overnight and washed with PBST. Goat anti-rabbit IgG (1:5000; Elabscience, Wuhan, China) or goat anti-mouse IgG (1:5000; Biogot, Nanjing, China) labeled with horseradish peroxidase was added and incubated at room temperature for 1 h and washed with PBST. Following thrice washing, the membranes were detected using a Western blot ECL kit (EpiZyme, Shanghai, China).
6
Western Blot Analysis of Corneal Proteins
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Mice corneas (n = 6/group/time) were harvested at 3 days p.i. Protein extraction and concentration determination were performed as described previously.4 (link) The protein was separated on 12% acrylamide SDS-PAGE and transferred to polyvinylidene difluoride (PVDF; Solarbio) membrane. Membranes were washed in PBS containing 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) (PBST) three times. After being blocked with blocking buffer (Beyotime, Jiangsu, China) at 37°C for 2 hours, the membrane was incubated with antibodies to GAPDH (1:1000; Elabscience, Wuhan, China), CXCL-1 (1:1000; Affinity Biosciences, Jiangsu, China), LC3B (1:1000; Cell Signaling Technology, Danvers, MA, USA), SQSTM1/p62 (1:1000; Cell Signaling Technology), Beclin-1 (1:1000; Cell Signaling Technology), LAMP-1 (1:1000; Cell Signaling Technology), IL-1β (1:1000; NOVUS, Littleton, CO, USA), IL-18 (1:500; Abcam, Cambridge, MA, USA), HMGB1 (1:1000; Abcam), TNF-α (1:2000; Proteintech Group, Wuhan, China), or IL-10 (1:1000; Abcam) at 4°C overnight. Membranes were washed in PBS containing 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) (PBST) three times. Membranes were incubated with corresponding peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37°C for 1 hour. Membranes were developed by chemiluminescence (ECL; Thermo Fisher Scientific, Waltham, MA, USA).
7
Western Blot Analysis of GPR43 Expression
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The cultured CD4+ T cells under Th17/Treg differentiation conditions were lysed in RIPA lysate (Thermo Scientific, USA) plus protease inhibitors (Beyotime, China). Proteins were subjected to electrophoresis separation and then transferred to polyvinylidene fluoride membranes. The membranes were incubated with 5% milk for 1 h, followed by incubating with the GPR43 antibody (1:800, Genetex) overnight at 4°C. GAPDH (1:5,000, Elabscience) was applied as a loading control. The membranes were washed in TBST, incubated with a corresponding secondary antibody (anti-rabbit or anti-mouse IgG, 1:5,000, Elabscience) for 1 h, and washed again by TBST. The protein bands were visualized by an enhanced chemiluminescence (ECL) system (ChemiScope 5600; Hengmei Technology, China).
8
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Cells were washed twice with PBS and lysed in RIPA buffer (Beyotime, Haimen, China) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). Protein lysates were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and incubated with primary antibodies for 24 h at 4°C. Membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, and immunoreactive bands were visualized with ECL chromogenic substrate (ComWin Biotech Co., Beijing, China). GAPDH was used as an internal control. Primary antibodies were as follows: E-cadherin (Ab133597; Abcam, Cambridge, UK), vimentin (CBL202; EMD Millipore, Billerica, MA, USA), β-catenin (Ab32572; Abcam), c-myc (Ab32072; Abcam), and GAPDH (ENM0215; Elabscience, Bethesda, MD, USA).
9
Western Blot Analysis of Apoptosis Markers
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Total proteins were collected from cells using RIPA lysis buffer (Solarbio, Beijing, China) with protease inhibitors (Solarbio, Beijing, China) on ice and quantified by a bicinchoninic acid (BCA) assay (Solarbio, Beijing, China). Equal amounts of protein were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Solarbio, Beijing, China). The protein bands were blocked in freshly prepared 5% skim milk, followed by blotting with antibodies against Bcl-2 (1 : 1000; Abcam, Cambridge, UK), Bax (1 : 1000; Abcam, Cambridge, UK), caspase 3 (1 : 1000; Cell Signaling Technology, USA), cleaved-caspase 3 (1 : 1000; Cell Signaling Technology, USA), caspase 9 (1 : 1000; Cell Signaling Technology, USA) and GAPDH (internal reference gene, 1 : 1000; Elabscience Biotechnology Co., Ltd., China) at 4°C overnight. Then, peroxidase-conjugated secondary antibodies (1 : 5000; Elabscience Biotechnology Co., Ltd., China) were used to incubate the membranes. Subsequently, electrochemiluminescence (ECL) solution was applied to detect each band with a chemiluminescence imaging system. The gray value of the protein bands was measured by ImageJ software and normalized to the expression of GAPDH.
10
Verification of Protein Expression
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We verified the protein expression of MMP14, PCDH7, and LAMP3 using immunohistochemical staining images from the HPA database (https://www.proteinatlas.org/ ). Moreover, we also verified the differential expression of CCL20, BTG2, IL7R, and TLR2 between normal bronchial epithelial cell line (16-HBS) and lung adenocarcinoma cell line (A549) by utilizing Western blotting. 16-HBS and A549 cell lines were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Protein concentrations were evaluated through the BCA Assay kit (CWBIO). Western blotting was performed as seen previously (Wang et al., 2020 (link)). Briefly, 15 ug proteins were loaded onto 8–15% polyacrylamide gels. After transfer, the NC membranes (Boster) were blocked with 5% skimmed milk at room temperature for 2 h. Next, membranes were incubated with primary antibodies [CCL20 (Affinity, 1:300), BTG2 (proteintech, 1:300), IL7R (Affinity, 1:300), TLR2 (proteintech, 1:1000)], and GAPDH (Elabscience, 1:3000) for 2 h at room temperature and then at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (CWBIO, 1:1000) at room temperature for 2 h. Finally, the bands were exposed with ECL reagents (CWBIO). ImageJ software was applied to perform the gray value analysis.
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