Whole cell lysates were prepared and the immunoblotting was done as previously described [3 (link)] in Triton X 100-containing lysis buffer (50 mM Tris–HCl, pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF). For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA). As secondary antibodies, IRDye® 800CW Donkey anti-Mouse IgG or IRDye® 680RD Donkey anti-Rabbit IgG (LI-COR, Lincoln, NE, USA) were used at 1:10 000 dilution.
Ppi3k p85 p55
Manufactured by Cell Signaling Technology
Sourced in United States
The PPI3K p85/p55 is a lab equipment product manufactured by Cell Signaling Technology. It is a key component in the phosphoinositide 3-kinase (PI3K) signaling pathway, which plays a crucial role in various cellular processes. The product consists of the p85 and p55 regulatory subunits of PI3K, which regulate the catalytic activity of the enzyme.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (3 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Pi3k p85α, by Cell Signaling Technology (1 mentions)
Anti p tyr 4g10, by Merck Group (1 mentions)
Irdye 680rd donkey anti rabbit igg, by LI COR (1 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Mek1 2, by Cell Signaling Technology (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Stat6, by Cell Signaling Technology (1 mentions)
Cell culture media, by Thermo Fisher Scientific (1 mentions)
Arginase assay kit, by BioAssay Systems (1 mentions)
Anti ym1 antibody, by STEMCELL (1 mentions)
Anti arginase 1 antibody, by BD (1 mentions)
4 protocols using ppi3k p85 p55
1
Immunoblotting Analysis of Cellular Signaling
2
Immunofluorescence Analysis of Macrophage Markers
3
Western Blot Analysis of PI3K/AKT Pathway
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Protein was extracted using RIPA buffer (Sigma-Aldrich). 25ug/well protein lysate was loaded onto an 8%–12% SDS-PAGE gel and was electro-transferred to a PVDF (polyvinylidene difluoride) membrane. The membrane was blocked in 5% non-fat milk for 30 min and was incubated with primary antibodies overnight at 4°C. The next day, we washed the membrane with TBST buffer and incubated the membrane with secondary antibodies. The membrane was developed by ChemiDoc MP System (Bio-Rad, USA). The antibodies used in the present study: p-AKT (Ser-473): #4060S, AKT (C67E7): #4691S, p-PI3K (p85/p55): #17366S, PI3K (p85): #4257S, β-ACTIN: #3700S were purchased from Cell Signaling Technology. Results were analyzed using ImageJ software (NIH, Version 1.53).
4
Western Blot Analysis of Autophagy
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The tumor masses and 143B cells cultured with or without TIIA were harvested and total cell protein was extracted using whole-cell lysis buffer. The protein concentrations were determined by the Bradford method (Bio-Rad, CA, USA). Samples with equal amount of protein were subjected to 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidenedifluoride (Millipore, Bedford, MA, USA) membrane. The membrane was incubated at room temperature in blocking solution (5% nonfat milk) for 1 h followed by incubation for 2 h in blocking solution containing an appropriate dilution of anti-LC3B, -P62, -SESN2, -HIF-1α, -castalase, -MnSOD, -GPX1, -Beclin 1, -ATG5, -ATG7, -JNK1, -p-SAPK/JNK, -c-Jun, -p-c-Jun, -PI3K 110λ, -p-PI3K p85/p55, -PI3K class III, -Akt, -p-Akt, -AMPK, -p-AMPK, -mTOR, and -p-mTOR antibody (Cell Signaling Technology, USA). After washing, blots were then probed with appropriate secondary horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and detected by an ECL detection system (Millipore) and scanned by MultiGel-21 (Top Bio, Taiwan). β-actin served as internal control.
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