Our previously published western blotting assay procedure was used to evaluate endogenous MAP1B expression and the MAP1B-knockdown efficiency in RTCC1 and J82 cell lines using primary antibodies against MAP1B (1:500, clone AA6; Millipore, Beverly, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GADPH) (6C5, 1:10,000; Millipore, Beverly, MA, USA). Cell lysates with 25 μg of protein were separated using a 4% to 12% gradient NuPAGE gel (Invitrogen, Carlsbad, CA, USA), then transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK) for the immobilization of proteins. Membranes were incubated with tris-buffered saline containing Tween 20 (TBST) buffer and 5% skimmed milk at room temperature for one hour for blocking, followed by exposure to primary antibodies at 4 °C overnight against MAP1B (1:500, clone AA6; Millipore, Beverly, MA, USA) using GADPH as a loading control (6C5, 1:10,000; Millipore, Beverly, MA, USA). Membranes were incubated with the secondary antibody at room temperature for 1.5 h, and proteins were detected using a chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK).
Gadph
Manufactured by Merck Group
Sourced in United States
GADPH is a lab equipment product that serves as an enzyme involved in the glycolysis pathway, catalyzing the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. It is commonly used in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Chemiluminescence system, by Cytiva (1 mentions)
Clone aa6, by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Vegfr 3, by Merck Group (1 mentions)
Rhvegf c, by R&D Systems (1 mentions)
Rhvegfa, by R&D Systems (1 mentions)
P vegfr2, by Cell Signaling Technology (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Nylon membrane, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail 1, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
β catenin, by Merck Group (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Axin2, by Cell Signaling Technology (1 mentions)
Hpv16e6, by Abcam (1 mentions)
Pd l1, by Abcam (1 mentions)
20 protocols using gadph
1
Quantifying MAP1B Expression by Western Blot
2
Modulation of VEGFR Signaling by AD0157
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Serum-starved subconfluent LEC cultures were incubated in the presence or absence of AD0157 for 2 h and then challenged for 30 additional minutes with rhVEGF-C (400 ng/mL), rhVEGF-C156S (500 ng/mL; R&D Systems), or rhVEGF-A (100 ng/mL; R&D Systems). Cell lysates were analyzed by SDS-PAGE, electrotransferred to nitrocellulose membranes (GE Healthcare Life Sciences), and blots were probed with primary antibodies against pVEGFR-3 (1/1000; Cell Application), VEGFR-3 (1/1000; Millipore), pVEGFR-2, VEGFR-2, pERK1/2, ERK1/2, pAkt, Akt, (1/1000; Cell Signaling), and GADPH (1/2000; Millipore). Detection was carried out with chemiluminescence system ECL Western Blotting Substrate (Pierce), and bands were quantified and expressed as phosphorylated protein/total protein ratio.
3
Western Blot Analysis of Cell Signaling Proteins
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Total cell protein lysates was extracted from cells in RIPA buffer (25 mM Tris (pH 7.8), 2 mM EDTA, 20% glycerol, 0.1% Nonidet P-40 (NP-40), 1 mM dithiothreitol). All protein extraction buffers were supplemented with MiniComplete protease inhibitor cocktail (Roche Diagnostic) and phosphatase inhibitor cocktail 1 (Sigma-Aldrich). The protein concentration was measured using Bradford reagent (Bio-Rad, Sundbyberg, Sweden). Equal quantities were separated by SDS-PAGE, transferred to nylon membranes (Millipore, Sundbyberg, Sweden), and probed with antibodies against active β-catenin (1:1,000, clone 8E7, Millipore, Solna Sweden), Axin2 (1:1,000, Cell Signaling Technology, Beverly, MA, USA), MGMT (1:1,000, Cell Signaling Technology), GADPH (1:10,000, Millipore) and β-actin (1:5,000, Cell Signaling Technology). Anti-rabbit IgG or anti-mouse IgG, conjugated with horseradish peroxidase (Pharmacia Biosciences, Uppsala, Sweden), were used as secondary antibodies. Pierce Super Signal (Pierce, Rockford, IL, USA) was used for chemiluminescent detection. MGMT recombinant protein served as positive control (Assay Designs, Ann Arbor, MI, USA). Quantification of blots were done with densitometry measurements in ImageJ32 (link).
4
Immunoblot analysis of HPV and PD-L1
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Whole cell lysates were mixed with Laemmli loading buffer, boiled, separated by SDS–PAGE, and transferred to a nitrocellulose membrane. Subsequently, immunoblot analyses were performed using antibodies specific to HPV16E6 (abcam, Cambridge, MA, USA), HPV16E7 (CERVIMAX, Vienna, Austria), HIF1α (Cell Signaling, Danvers, MA, USA), PD-L1 (abcam), or GADPH (Millipore).
5
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed in RIPA buffer containing protease inhibitor (Beyotime, Shanghai,China). Protein samples were separated by SDS-PAGE,then transferred to polyvinylidene uoride (PVDF) membrane. The membranes were incubated with E-cadherin (Abcam, shanghai, China), N-cadherin (Abcam, China), Vimentin (Abcam), and GADPH (Bioss, Beijing, China) primary antibodies. Protein expression was assessed by ECL chemiluminescent regents (millipore, USA) and quanti ed by densitometry (Image J software) and normalized to the corresponding GADPH bands.
6
Immunoblotting Assay for STAT5 Activation
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Cells incubated with IL-2 with/without 20 μg/ml MBL for indicated time were harvested. The protein lysates were prepared using RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, and pH 7.4), subsequently separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in TBS containing 0.05% Tween-20 (TBST) and 5% BSA for 1 h at RT and incubated overnight at 4°C with the antibodies against STAT5 (clone 89/Stat5), phospho-STAT5 (clone 47/Stat5 (pY694)) (BD Pharmingen, San Diego, CA, USA), or GADPH (Sigma-Aldrich, St. Louis, MO, USA).
7
Immunoblotting for Protein Detection
8
Western Blot Analysis of Protein Expression
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Proteins were extracted with RIPA buffer containing an inhibitor cocktail (Roche, Sussex, UK) and protein concentration was determined using a Bradford dye-based assay (Bio-Rad, Hemel Hempstead, UK). Total protein (30 μg) was subjected to SDS–PAGE followed by immunoblotting with appropriate antibodies at the recommended dilutions. The blots were then incubated with peroxidase-linked secondary antibodies followed by enhanced-chemiluminescent detection using the Super Signal Chemiluminescence Kit (Thermo scientific). The following antibodies were used: PGC-1α (rabbit, 1 : 1000, Abcam, Cambridge, UK), MEF2 (rabbit, 1 : 500, Santa Cruz, Middlesex, UK), Phospho-S6 Ribosomal Protein (Ser235/236) (rabbit, 1 : 100, Cell Signaling Technology, UK), S6 Ribosomal Protein (5G10) (Rabbit, 1 : 100, Cell Signaling Technology), TFAM (rabbit, 1 : 100, Sigma, UK), NRF1 (rabbit, 1 : 500, Life Span Bioscience, Nottingham, UK), Cleaved caspase-3 (rabbit, 1 :1 000, Cell Signaling), GAD67 (mouse, 1 : 1000, Millipore, Watford, UK), MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (mouse, 1 : 250, Abcam, UK), LC3B (rabbit, 1 : 1000, Sigma), β-Tubulin (rabbit, 1 : 1000, Santa Cruz), Synapsin (1 : 1000, Synaptic System, Goettingen, Germany), Actin (1 : 5000, Sigma), Cyclophillin D (1 : 1000, Abcam), GLS2 (1 : 100, Abcam), PFKp (1 : 500, Abcam) and GADPH (mouse, 1 : 10 000, Sigma).
9
Protein Extraction and Analysis Workflow
10
VEGF Protein Quantification Protocol
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The tissue proteins extracted were determined for concentration by following the instructions on the BCA Kit (Beyotime, Beijing). After the addition of loading buffer, proteins were boiled at 95°C for 10 min and 30 μg samples were loaded in each well. Next, 10% polyacrylamide gel electrophoresis (PAGE) was applied to isolate proteins, which were transferred to polyvinylidene fluoride (PVDF) membrane and closed in 5% bovine serum albumin (BSA) for 1 hr at room temperature. Then, primary antibody for VEGF (diluted by 1:500) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) and GADPH (diluted by 1:1000) (Sigma-Aldrich Chemical Company, St Louis MO, USA) were added for overnight reaction at 4°C. Next day, the membrane was washed with TBS Tween 20 (TBST) for 3 times/5 min before adding corresponding secondary antibody for 1 hr of incubation. After that, the membrane was washed again for 3 times/5 min, followed by development using chemiluminescence method, and GAPDH was the loading control.
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