Primary antibodies (Abs) -- OGG1 Ab (Cat # ab124741) and subtype-specific HRAS Ab (Cat# 1521–1) were purchased from Epitomics (Burlingame, CA, USA). pan-RAS Ab (Cat# 05–1072) was from Millipore (Billerica, MA) and KRAS (Cat # sc-30), NRAS (Cat# sc-3), RelA (Cat# sc-372), and p-RelA (Ser276, Cat # sc-101749) Abs from Santa Cruz Biotechnology (Dallas, TX, USA). The following Abs were purchased from Cell Signaling Technologies (Danvers MA, USA): RAF-1 (Cat# 9422), phosphorylated (p)-RAF-1 (Ser338 Cat# 9427), ERK 1,2 (Cat # 4695), p-ERK 1,2 (Thr202/Tyr204, Cat# 9101), MEK 1,2 (Cat# 9122), p-MEK1,2 (Ser217/221 Cat# 9154), MSK1 (Cat# 3489), p-MSK1 (Ser376) Cat# 9591), PI3K(p85, Cat #4292), p-PI3K (p85/Tyr458; p55/Tyr199, Cat# 4228), AKT (Cat #9272), p-AKT (Ser473, Cat# 4058), IKKα (Cat#2682), IKKβ (Cat#2370), p-IKKα/β (Ser176/180, Cat #2697), IκBα (Cat# 4814), and p-IκBα (Ser32/36 Cat# 9246). Secondary Abs, e.g., goat anti-rabbit, rabbit anti mouse and donkey anti-mouse, were from GE Healthcare (Pittsburgh, PA, USA).
P pi3k p85 tyr458 p55 tyr199
Manufactured by Cell Signaling Technology
Sourced in United States
The P-PI3K p85 (Tyr458)/p55 (Tyr199) is a lab equipment product that detects the phosphorylation of the p85 and p55 regulatory subunits of PI3-kinase at the indicated tyrosine residues. This product is used to measure the activation of the PI3-kinase signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Pi3k p85, by Cell Signaling Technology (3 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P msk1, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
Raf 1, by Cell Signaling Technology (1 mentions)
P rela, by Santa Cruz Biotechnology (1 mentions)
Mek1 2, by Cell Signaling Technology (1 mentions)
P ikkα β, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
M pertm mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
5 protocols using p pi3k p85 tyr458 p55 tyr199
1
Comprehensive Signaling Pathway Analysis
2
Protein Extraction and Western Blot Analysis
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Protein lysates were extracted using M-PERTM Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) containing HaltTM Protease Inhibitor cocktail and EDTA (Life Technologies, USA). Protein concentration was determined by the Microtiter Bio-Rad Protein Assay solution (Bio-Rad Laboratories, CA, USA). 30 μg of proteins were loaded onto SDS-PAGE gels, and standard western blot analysis was performed. The following antibodies were used: GRAMD1B (Abcam, ab121286), Rac1/RhoA/Cdc42 (Cell Biolabs, Inc., #STA-404), p-STAT3 (Tyr705) (Cell Signaling Technology, #9145), STAT3 (Cell Signaling Technology, #12640), p-JAK2 (Tyr1007/1008) (Cell Signaling Technology, #3771), JAK2 (Cell Signaling Technology, #3230), p-Akt (Ser473) (Cell Signaling Technology, #4060), Akt (Cell Signaling Technology, #4691), p-PI3K p85 (Tyr458)/p55 (Tyr199) (Cell Signaling Technology, #4228), PI3K p85 (Cell Signaling Technology, #4257) and β-actin (Sigma- Aldrich, A2228).
3
Comprehensive Western Blot Analysis Protocol
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We carried out Western Blot analysis as described previously [7 (link)]. Membranes were incubated with following primary antibodies for overnight at 4 °C: SEMA5A (Invitrogen; 1:1000), anti-E-cadherin (E-cad) (HECD1; 1:100), anti-N-cadherin (N-cad) (1:100), anti-β-catenin (1:200), anti-SNAIL (Abcam; 1:500), anti-GAPDH (Santa Cruz, 1:1000) and anti-β-actin (Sigma, 1;1000). PI3K (Sigma, 1:1000), p-PI3K (p85Tyr458-p55Tyr199, Cell Signaling, 1;750), AKT (Cell Signaling 1:1000), p-AKT (Ser473, Cell Signaling, 1:1000), GSK3β (Cell Signaling, 1:1000) p-GSK3β (Ser9, Cell Signaling 1:1000). Anti-E-cad, anti-N-cad, and anti-β-catenin were generous gift from Dr. Keith Johnson, University of Nebraska Medical Center; UNMC, Omaha, NE, USA. The membrane was incubated with secondary horseradish peroxidase antibody (mouse (Sigma), 1:5000, rabbit (Thermo Scientific); 1:5000) for an hour at room temperature. We utilized NIH Image J Software for quantification of the intensity of the bands, of our protein of interest and their respective loading control. We also normalized the bands to the Control cells used in the study.
4
Western Blot Analysis of TGF-β1 Signaling
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Total protein concentration of homogenate was measured using a BCA protein assay kit (Beyotime Biotech Inc., Shanghai, China) according to the manufacturer's protocol and was equalized before electrophoresis. Briefly, 40 μg of the proteins in the supernatant was separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking at room temperature for 1 h with 5% nonfat dry milk, the membranes incubated with antibodies TGF-β1 (1 : 500 dilution, Wanleibio, Shijiazhuang, China), PTEN (1 : 400 dilution, Wanleibio, Shijiazhuang, China), PI3K p85 (1 : 1000 dilution, Cell Signaling Technology, USA), p-PI3K p85 (Tyr458)/p55 (Tyr199) (1 : 1000 dilution, Cell Signaling Technology, USA), and Akt and p-Akt (Ser473) (1 : 1000 dilution, Cell Signaling Technology, USA) overnight at 4°C. After washing with TBST, the membranes were incubated with IgG-HRP (1 : 5000 dilution, Wanleibio, Shijiazhuang, China) for 1 h at room temperature. The membranes were developed with enhanced chemiluminescence using ECL reagents (Beyotime Biotech Inc., Shanghai, China) and visualized using a digital imaging system (Bio-Rad Laboratories, Inc., USA). The blots were quantitated by densitometric analysis using NIH ImageJ software. The data were normalized on the basis of GAPDH level.
5
Molecular Signaling Pathway Analysis
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Western blot analysis was performed using antibodies against Mcl-1, JNK (56G8), pJNK (81E11), pMEK (41G9), MEK (D1A4), pERK1/2 (197G2), ERK1/2 (137F5), pAKT (S473) (193H12), pAKT (T308) (244F9), AKT, pS6 (Ser235/236), S6, p4EBP-1 (Thr70), 4EBP-1, pPI3K p85 (Tyr458)/p55(Tyr199) (Cell Signaling Technology, Danvers, MA, USA), PI3K p110 delta (Novus Biologicals), caspase-3, p21, IER3 (Santa Cruz Biotechnologies, San Diego, CA, USA), and poly(ADP-ribose) polymerase (PARP) (BD).
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