The cells were lysed in lysis buffer (Beyotime, Shanghai, China) for 15 min on ice, then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking the membrane with 5% low-fat milk for 2 h at room temperature, the membrane was probed with the appropriate primary antibodies for 2 h at room temperature. HRP-conjugated goat anti-mouse and -rabbit IgG (H-L) secondary antibodies were treated with membranes (1:1000; Beyotime, Shanghai, China). The Tanon 5200 chemiluminescence imaging system was utilized to detect bound proteins (Tanon, Shanghai, China). The bands were measured using ImageJ (Version 5.1) and normalized to β-actin levels for quantitative analysis. The primary antibodies used in Western blotting (WB) (anti-β--actin, anti-ERK1/2, anti-phospho-ERK1/2, anti-PI3K, anti-phospho-PI3K, anti-AKT and anti-phospho-AKT) were purchased from Abcam.
Anti phospho pi3k
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-phospho-PI3K is a primary antibody that recognizes the phosphorylated form of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K). It is used in research applications to detect and quantify the levels of activated PI3K signaling in cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt, by Abcam (5 mentions)
Anti pi3k, by Abcam (4 mentions)
Anti akt, by Abcam (3 mentions)
Anti erk1 2, by Abcam (2 mentions)
Anti phospho erk1 2, by Abcam (2 mentions)
Lysis buffer, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
Nitrocellulose filter membrane, by Bio-Rad (1 mentions)
Anti integrin β1, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Coomassie protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erbb3, by Abcam (1 mentions)
5 protocols using anti phospho pi3k
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of Integrin β1, PI3K, and Akt
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Cells were lysed in cold RIPA lysis buffer supplemented with Protease Inhibitors cocktail (CST Inc., USA). The protein concentration was quantified using BCA protein assay kit (Beyotime, China) following the manufacturer’s introduction. Twenty micrograms of protein were mixed with 1× loading buffer and boiled for 5 mins at 100°C and were resolved by 8% or 10% SDS-PAGE and transferred to the nitrocellulose filter membrane (Bio-Rad). Target protein was detected using specific primary antibody, including: anti-Integrin β1 (1:1000; Abcam, Cambridge, UK), anti-PI3K (1:1000; Abcam), anti-Akt (1:1000; Abcam), anti-phospho-PI3K (1:1000; Abcam), anti-phospho-Akt (1:1000; Abcam). All experiments were repeated three independent times and the target protein level was quantified by ImageJ and normalized to internal control.
3
Western Blot Analysis of Apoptosis and Signaling Pathways
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The protein concentration was determined using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, United States) (Yi et al., 2016 (link)). Equal amounts of protein samples were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States) (Gu et al., 2015 (link)). The membranes were blocked in 5% nonfat dry milk for 2 h and incubated with primary antibody anti-Bcl-2 (1:1000; Abcam), anti-Bax (1:1000; Abcam), anti-caspase-3 (1:1000; Abcam), anti-cleaved caspase-3 (1:1000; Abcam), anti-phospho ERK1/2 (1:1000; Abcam), anti-ERK1/2 (1:1000; Abcam), anti-PI3K (1:1000; Abcam), anti-phospho PI3K (1:1000; Abcam), anti-phospho AKT (1:1000; Abcam), anti-AKT (1:1000;Abcam), and anti-GAPDH (1:5000; Abcam) overnight at 4°C. After three washes with Tris-buffered saline with 0.1% Tween-20 (TBST), HRP-conjugated secondary antibodies (Pierce) were used to incubate the membranes for 1 h at room temperature. The membranes were developed using a chemiluminescence reagent (Roche) after three times washed in TBST and exposed to Kodak exposure films. GAPDH served as an internal control in order to make normalization and relative quantitative analysis of target protein expression (Feng et al., 2016 (link)).
4
Western Blot Analysis of AKT and PI3K
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Samples were prepared from whole-cell pellets followed by lysis with Nonidet-P40
(NP40). Total protein was quantified using Coomassie Protein Assay Reagent
(Pierce ThermoFisher Scientific, Rockford, IL, USA). Equal amounts of protein
(30 µg) were separated by 10% SDS–PAGE and electro-transferred onto the PVDF
membrane. Primary antibodies used were: anti-AKT (Cell Signaling Technology, CST
4691S), anti-phospho-AKT (CST 4060S), anti-PI3K (CST 4257P), anti-phospho-PI3K
(CST 4228P), anti-ERBB3 (Abcam ab255607) and anti-β-actin (Sigma A5441). The
secondary HRP conjugated antibodies used were anti-rabbit IgG (CST 7074) and
anti-mouse IgG (CST 7076).
(NP40). Total protein was quantified using Coomassie Protein Assay Reagent
(Pierce ThermoFisher Scientific, Rockford, IL, USA). Equal amounts of protein
(30 µg) were separated by 10% SDS–PAGE and electro-transferred onto the PVDF
membrane. Primary antibodies used were: anti-AKT (Cell Signaling Technology, CST
4691S), anti-phospho-AKT (CST 4060S), anti-PI3K (CST 4257P), anti-phospho-PI3K
(CST 4228P), anti-ERBB3 (Abcam ab255607) and anti-β-actin (Sigma A5441). The
secondary HRP conjugated antibodies used were anti-rabbit IgG (CST 7074) and
anti-mouse IgG (CST 7076).
5
Protein Extraction and Western Blot Analysis
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For the extraction of total protein lysates, RIPA lysis buffer (Beyotime, Nantong, China) containing protease inhibitor was used. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Subsequently, the samples were transferred onto a polyvinylidene fluoride membrane, which was then blocked with 5 % BSA for 1.5 h. The membrane was subsequently probed with primary antibodies, including anti‐CSMD2 (NBP2-92141; Novus, Centennial, CO, USA), β-Tubulin (#10094-1-AP; Proteintech, Wuhan, China), anti‐phospho‐AKT (phosphorylated at Ser473; Abcam, Cambridge, UK), anti‐AKT (#4685; CST, Danvers, MA, USA), anti‐PI3K (#3011S; CST), and anti‐phospho‐PI3K (phosphorylated at Y607; Abcam). The samples were incubated overnight at 4 °C, followed by washing in Tris‐buffered saline buffer with Tween‐20 four times for 8 min. Then, horseradish peroxidase-conjugated secondary antibodies were applied for 1.5 h. After washing the membrane, protein level bands were visualized using an enhanced Electro‐Chemi‐Luminescence kit (Beyotime) on a ChemiDoc MP imaging system (Bio‐Rad, Hercules, CA, USA). The relative quantity of proteins was determined as the ratio of the protein of interest to an internal reference.
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