Western blotting was performed as previously described [25 (link)] using antibodies against RhoB (1:500; Proteintech, USA), phospho-Akt 308, phospho-Akt 473 and Akt (1:1,000; Cell Signaling Technology, USA), phospho-PI3K (1:1,000; Abcam, USA), PI3K (1:1,000; Cell Signaling Technology, USA), P-glycoprotein (P-gp, also known as ABCB1) (1:500; Proteintech, USA), breast cancer resistance protein (BCRP, also known as ABCG2) (1:500; Proteintech, USA), multidrug resistance-associated protein 1 (MRP1, also known as ABCC1) (1:500; Abcam, USA) and β-actin (1:5,000; Cell Signaling Technology, USA). The secondary antibody used is horseradish peroxidase-conjugated antibody (anti-rabbit; 1:5,000; Cell Signaling Technology). Protein levels were normalized to the endogenous control β-actin.
Phospho pi3k
Manufactured by Abcam
Sourced in United States
Phospho-PI3K is a lab equipment product that detects and quantifies the phosphorylation of the phosphoinositide 3-kinase (PI3K) enzyme. PI3K plays a crucial role in various cellular processes, including cell growth, proliferation, and survival. This product allows researchers to measure the activation state of PI3K, which is important for understanding signaling pathways and cellular responses in a variety of experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Abcam (2 mentions)
Phospho akt 308, by Cell Signaling Technology (1 mentions)
Phospho akt473, by Cell Signaling Technology (1 mentions)
Abcc1, by Abcam (1 mentions)
Ferritin, by Abcam (1 mentions)
Mem pertm plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
T pi3k, by Abcam (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Active β catenin, by Cell Signaling Technology (1 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
β actin lk ab008 100, by Liankebio (1 mentions)
Znf545, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
7 protocols using phospho pi3k
1
Quantitative Western Blotting of Akt Pathway
2
Protein Extraction and Western Blot Analysis
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Cells were lysed by RIPA. After being shocked and ice-bath for 30 min, cells were centrifuged for 20 min at 22,000 rpm at 4°C. The supernatants were collected. Cell membrane protein and cytoplasmic protein were separated using Mem-PERTM Plus Membrane Protein Extraction Kit (Thermo). The concentration of protein was accessed using BCA. 20–40 μg protein was separated by 8–12.5% SDS-PAGE, then transferred to polyvinylidene fluoride membrane (Immobilon-P) using Trans-Blot Turbo Transfer System (Bio-Rad). DMT1, IRP1, IRP2, TfR1, TfR2, ferritin, T-PI3K, phospho-PI3K antibodies were obtained from Abcam. T-STAT3 (0.1 mg/ml), phospho-STAT3 tyr705 (1 mg/ml) were acquired from R&D. T-ERK, phospho-ERK, pan-AKT, phospho-AKT (1:1000) were obtained from Cell signaling technology. GAPDH and β-actin antibodies were purchased from Biotime. After being cultured with primary antibodies overnight at 4 °C, proteins were cultured with the HRP-labeled secondary antibody (KPL). Proteins were visualized using enhanced chemiluminescence (Pierce). All experiments were performed in triplicate.
3
Western Blot Analysis of Apoptosis and Signaling
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A total of 40 mg of protein lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The primary antibodies were against cleaved caspase-3 (12742; Cell Signaling Technology, Danvers, MA, USA), cleaved poly(ADP-ribose) polymerase (PARP) (9541; Cell Signaling Technology), BAX (5023; Cell Signaling Technology), ZNF545 (sc-102235; Santa Cruz Biotechnology, Santa Cruz, CA, USA), PI3K (ab125571; Abcam, Cambridge, UK), phospho-PI3K (Abcam), Akt (4691; Cell Signaling Technology), phospho-Akt (ser473) (4060; Cell Signaling Technology), phospho-GSK3β (ser9) (9323; Cell Signaling Technology), β-catenin (2677; Cell Signaling), active β-catenin (4270; Cell Signaling), c-Myc (1472-1; Epitomic), cyclin D1 (2261; Epitomics), β-actin (LK-ab008-100; Liankebio, China), and Flag (F3165; Sigma-Aldrich).
4
Western Blotting Analysis of PI3K/Akt Pathway
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Western blotting was performed as described in our previous study [11 (link),13 (link)]. Meg-01 cells were lysed with protein lysis buffer (P0013, Beyotime Biotechnology, Haimen, China) containing a protease inhibitor cocktail (539131, Millipore, Billerica, MA, USA) and phosphatase inhibitor cocktail (K1012, APExBIO, Houston, TX, USA). The samples (20 μg) were then separated by 8% SDS-PAGE gel and transferred onto PVDF membranes. The PDVF membranes were incubated with primary antibodies at 4 °C for 12 h. After incubating with the appropriate HRP-conjugated secondary antibodies (7076, 7074, Cell Signaling Technology, Danvers, MA, USA), blots were detected with chemiluminescent HRP substrate (P90719, Millipore, Billerica, MA, USA) using the ChemiDoc XRS+ system (BIO-RAD, Hercules, CA, USA). Primary antibodies against the following antigens were listed: PI3K (4257, Cell Signaling Technology, Danvers, MA, USA), Phospho-PI3K (ab226842, Abcam, Cambridge, UK), Akt (4691, Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt (4056, Cell Signaling Technology, Danvers, MA, USA), and β-actin (66009-1, Proteintech, Rosemont, IL, USA).
5
Protein Expression Analysis in Cells
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Proteins were extracted from tissue samples or cells. Total protein concentration was determined using a bicinchoninic acid (BCA) kit for protein determination (Takara). The samples were resolved in 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Immunoblots were probed with primary antibodies overnight at 4°C. The following antibodies used were from Abcam: H3R (1:1000), E-cadherin (1:10000), N-cadherin (1:5000), ZO-1 (1:50), vimentin (1:1000), phospho-PI3K (Tyr607)(1:500). The following antibodies were from Cell Signaling: phospho-Akt (Ser473) (1:1000), Akt (1:1000), phospho-ERK1/2 (Thr202/Tyr204)(1:1000), ERK1/2 (1:1000), phospho-MEK1/2 (Ser217/Ser221)(1:1000), MEK1/2 (1:1000), PI3K (1:1000) and β-actin (1:1000). All primary antibodies were used according to the instructions provided by the supplier. After that, the membrane was incubated with the corresponding HRP-linked anti-rabbit IgG antibody (1:2000, Cell signaling) at room temperature for 1 h. The blots were visualized using the ECL-Plus reagent (Millipore).
6
Synthesis and Identification of LXQ46
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LXQ46 (3,4-dibromo-5-(5-(4-(4-ethoxyphenoxy)phenyl)oxazol-2-yl)benzene-1,2-diol) was synthesized and identified by our lab (purity 98%). Primary antibodies used in this study: PTP1B (#133259) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PARP (#9532), Bcl-xL (#2764), Phospho-AMPK (Thr172, #2535), AMPK (#2793S), Phospho-Akt (Ser473, #4060), Akt (#4685), Phospho-ERK (Thr202/Tyr204, #4370S), ERK (#4696S), Phospho-PRAS40 (#2640) and Ki67 (#9027) were purchased from Cell Signaling Technology (Danvers, MA, USA). Bax (#32503), Bcl-2 (#32124), CDK2 (#32147), CDK4 (#108357), CDK6 (#124821), Cyclin D1(#134175), Phospho-Src (Tyr529, #32078), Src (#47405), Phospho-PI3K (#182651), PI3K (#86714), PRAS40 (#151719) were purchased from Abcam (Cambridge, MA, USA). Phospho-p70 S6 Kinase (Thr389/412, #3228) and p70 S6 Kinase (#6226), Phospho-PKM2 (Tyr105 #7771), PKM2 (#5234) were purchased from Affinity Biosciences (Cincinnati, OH, USA). β-actin antibody and all secondary antibodies were obtained from Proteintech Group (Wuhan, China).
7
Signaling Pathway Profiling in Cancer Cells
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TPC‐1 and CAL‐62 cells were treated with protein lysates containing protease inhibitors. Each group maintained the same concentration of total protein, and proteins were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes. The protein‐containing membranes were then treated with 5% fat‐free milk and primary antibodies (TBK1, MAZ, phospho‐PI3K [p‐PI3K], PI3K, phospho‐Akt [p‐Akt], Akt, phospho‐mTOR [p‐mTOR], and mTOR; Abcam). The membranes were then treated with a secondary antibody (Abcam) for 1 h. The proteins were imaged by an Enhanced Chemiluminescent Kit (Thermo Fisher Scientific) and analyzed by ImageJ (V1.50b; NIH).
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