Western blot assays were performed using established protocols with the following antibodies: phospho-Akt (Ser473) 1:1000 (Cell Signaling 9271), Total Akt 1:1000 (Cell Signaling 9272), phospho-MEK1/2 1:5000 (Cell Signaling 9154), Total MEK1/2 1:5000 (Cell Signaling 9122), phospho-FGF Receptor (Tyr653/654) 1:500 (Cell Signaling 3471), FGF Receptor 2 (D4L2V) 1:500 (Cell Signaling 23328), p44/42 MAPK (Erk1/2) 1:5000 (Cell Signaling 9101), Total MAPK 1:5000 (Cell Signaling 9102), pFRS2 1:1000 (Cell Signaling 3864), anti-FRS2 (abcam ab10425), phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) 1:1000 (Cell Signaling 4228), PI3 Kinase p85 (19H8) 1:000 (Cell Signaling 4257), β-actin 1:10000 (Cell Signaling 4967) and GAPDH 1:10000 (Santa Cruz sc-25778).
Phospho pi3 kinase p85 tyr458 p55 tyr199
Manufactured by Cell Signaling Technology
Sourced in United States
The Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) is an antibody product used to detect the phosphorylation of the p85 and p55 subunits of phosphoinositide 3-kinase (PI3K) at specific tyrosine residues. This antibody can be used in various applications such as western blotting to study the activation of the PI3K signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P44 42 mapk erk1 2, by Cell Signaling Technology (3 mentions)
Phospho akt ser473, by Cell Signaling Technology (3 mentions)
Total mapk, by Cell Signaling Technology (2 mentions)
Total mek1 2, by Cell Signaling Technology (2 mentions)
Phospho mek1 2, by Cell Signaling Technology (2 mentions)
Pi3 kinase p85 19h8, by Cell Signaling Technology (2 mentions)
Phospho fgf receptor tyr653 654, by Cell Signaling Technology (2 mentions)
Fgf receptor 2 d4l2v, by Cell Signaling Technology (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pfrs2, by Cell Signaling Technology (1 mentions)
Anti frs2, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Pi3 kinase p85, by Cell Signaling Technology (1 mentions)
Igf 1 receptor β, by Cell Signaling Technology (1 mentions)
Molecular imager fxtm system, by Bio-Rad (1 mentions)
Tcf8 zeb1, by Cell Signaling Technology (1 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
6 protocols using phospho pi3 kinase p85 tyr458 p55 tyr199
1
Western Blot Antibody Profiles
2
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following treatment with drugs, cells were lysed and whole cell fractions were isolated as previously described [17 (link)]. Proteins (10–30 μg) were separated by 10%–12% (w/v) polyacrylamide gel electrophoresis and electroblotted to PVDF membranes. After blocking with non-fat milk for 1 h, membranes were incubated overnight with the following primary antibodies (1:1000 dilution) from Cell Signaling Technology (Danvers, MA, USA): E-Cadherin (Cat #3195), N-Cadherin (Cat #13116), TCF8/ZEB1 (Cat #3396), β-Catenin (Cat #8480), Vimentin (Cat #5741), phospho-Akt (Ser473) (Cat #4060), Akt (Cat #9272), phospho-IGF-I Receptor β (Tyr1135/1136) (Cat #3024), IGF-I Receptor β (Cat #9750), phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cat 4228), PI3 Kinase p85 (Cat 4257), phospho-mTOR (Ser2448) (Cat #5536), mTOR (Cat #2983). β-Actin (Cat #8457) was used at the same time as a loading control. After incubation with the secondary antibody (HRP-conjugated; 1:5000 dilution) for 1 h, at room temperature, the conjugates were developed and visualized using a Molecular Imager FXTM System (BioRad; Hercules, CA, USA).
3
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from HepG2 cells using RIPA lysis (Beyotime) according to manufacturer’s protocol and were quantified using a bicinchoninic acid kit (Beyotime). A total of 30 µg proteins were loaded to 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk in Tris-buffered saline with Tween-20 (TBST), PVDF membrane was incubated with primary antibodies (phospho-PI3 Kinase p85Tyr458/p55Tyr199, phospho-AKTSer473, total-PI3K, total-AKT, Bcl-2, Bax, and glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling Technology) overnight. After that, the membrane was washed with TBST three times and incubated with a secondary antibody (1:5,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 2 hours at 37°C. Target proteins were detected using the enhanced chemiluminescence system (Millipore) and visualized with the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
4
Investigating AKT/PI3K Signaling Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MK-2206 2HCl was purchased from Selleck Chemicals (Houston, USA). GIPC1 and PDGFR-α and PDGFR-β antibodies were obtained from Proteintech Group, Inc. (Chicago, USA). The phospho-AKT (Ser473) antibody and phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) were purchased from Cell Signaling Technology, Inc. (Danvers, USA). The PI3Kinase p85α antibody was obtained from Abcam (Cambridge, UK).
5
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and lysed in RIPA lysis buffer (Beyotime, P0013C) supplemented with the complete protease inhibitor cocktail (Yeasen, 20124ES03) on ice for 10 min. After centrifugation at 20,000 × g for 10 min at 4 °C, supernatant was collected and protein concentrations were quantified with a Bradford Protein Assay Kit (Coolaber, SK1060). After adding loading buffer (Beyotime, P0015L) and boiling at 98 °C for 5 min, protein extracts were resolved on an SDS-PAGE gel and then transferred to a nitrocellulose membrane (Pall, 66485). The membrane was blocked with 5% skim milk at room temperature for 1 h, followed by incubation with primary antibodies at 4˚C overnight. The following antibodies were used at 1:1000 dilution: Phospho-Stat5 (Tyr694) (Cell Signaling Tech, 9359), Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cell Signaling Tech, 4228), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Tech, 9101), p44/42 MAPK (Erk1/2) (Cell Signaling Tech, 4695), and LSD1 (Santa Cruz Biotech, sc-53875). After washing the membranes with PBST (PBS, 0.1% Tween-20) three times, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (Biosharp, BL003A) or HRP-conjugated goat anti-mouse IgG (Biosharp, BL001A) diluted in 5% skimmed milk at room temperature for 1 h. After three washes with PBST, ECL was applied to the membranes and imaged using Tannon-5200.
6
Signaling Pathway Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot assays were carried out using established protocols and probed with the following antibodies: phospho-Akt (Ser473) 1:1000 (Cell Signaling 9271), Total Akt 1:1000 (Cell Signaling 9272), phospho-MEK1/2 1:5000 (Cell Signaling 9154), Total MEK1/2 1:5000 (Cell Signaling 9122), p44/42 MAPK (Erk1/2) 1:5000 (Cell Signaling 9101), Total MAPK 1:5000 (Cell Signaling 9102), phospho-FGF Receptor (Tyr653/654) 1:500 (Cell Signaling 3471), FGF Receptor 2 (D4L2V) 1:500 (Cell Signaling 23328), phospho-PLCγ1 (Tyr783) 1:1000 (Cell Signaling 14008), PLCγ1 (D9H10) 1:1000 (Cell Signaling 5690), phospho-FRS2-α (Tyr196) 1:1000 (Cell Signaling 3864), FRS2 1:1000 (abcam 10425), phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) 1:1000 (Cell Signaling 4228), PI3 Kinase p85 (19H8) 1:000 (Cell Signaling 4257), β-actin 1:10000 (Cell Signaling 4967).
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!