Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from Gibco. Earle's balanced salt solution (EBSS) and propranolol were purchased from Sigma‐Aldrich. Rapamycin (RAPA), 3‐methyladenine (3‐MA) and chloroquine (CQ) were obtained from MedChemExpress, and ZVAD‐FMK, necrostatin‐1, liproxstatin‐1, SB203580, SP600125, SC79 and 740Y‐P were purchased from SelleckChem. Then, these reagents were dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals or water and stored at −80°C. Primary antibodies against Akt, p‐Akt, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, Erk1/2 and p‐Erk1/2 were purchased from Cell Signalling Technology. Primary antibodies against alpha‐smooth muscle actin (α‐SMA), fibronectin (FN), LC3B, P62, p‐PI3K p85, PI3K p85, ATG9b, ATG9a, mTOR, p‐mTOR, ATG12, ATG5 and anti‐ubiquitin were procured from Abcam. Primary antibodies against beta‐actin, alpha‐tubulin, GAPDH and HRP‐conjugated secondary antibodies were obtained from Proteintech. DyLight 549‐conjugated and DyLight 488‐conjugated secondary antibodies were provided by Abbkine.
P pi3k p85
Manufactured by Abcam
Sourced in United Kingdom
p85 (PIK3R1) is the regulatory subunit of phosphoinositide 3-kinase (PI3K). It acts as an adaptor protein, binding to the catalytic p110 subunit (PI3K) and regulating its activity.
Automatically generated - may contain errors
Lab products found in correlation
P mtor, by Abcam (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Necrostatin 1, by Selleck Chemicals (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Propranolol, by Merck Group (1 mentions)
Atg9a, by Abcam (1 mentions)
Dylight 488 conjugated secondary antibodies, by Abbkine (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Anti ubiquitin, by Abcam (1 mentions)
Pi3k p85, by Abcam (1 mentions)
Z vad fmk, by Selleck Chemicals (1 mentions)
Earle s balanced salt solution ebss, by Merck Group (1 mentions)
Sp600125, by Selleck Chemicals (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Liproxstatin 1, by Selleck Chemicals (1 mentions)
Atg12, by Abcam (1 mentions)
3 protocols using p pi3k p85
1
Comprehensive Cell Signaling Reagents Protocol
2
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDCs were harvested at 24 h after stimulation with LPS and frozen at −80°C. Total protein was extracted using a lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were determined by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Protein (40 μg/well) was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST) for 2 h at room temperature, then incubated with primary antibodies (p-PI3K p85, p-Akt T308, p-mTOR, Abcam, Cambridge, UK) diluted in a blocking solution (1:1,000) overnight at 4°C, and finally treated with diluted horseradish peroxidase (HRP)-conjugated secondary antibody (1:50,000, BOSTER Biological Technology Co., Wuhan, Chine) at 37°C for 2 h. The immunoreactive bands were analyzed using the BandScan 5.0 (Glyko, Novato, CA, USA) gel imaging software.
3
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from PC12 cells using pre-cold RIPA buffer (Beyotime Institute of Biotechnology) including protease inhibitor. Total protein was quantified using a bicinchoninic acid kit (Sigma-Aldrich; Merck KGaA). Proteins (40 µg) were then separated via 10% SDS-PAGE, proteins were transferred onto PVDF membranes. The membranes were then blocked with 5% skimmed milk at room temperature for 1 h. The membranes were incubated overnight at 4˚C with anti-PDCD4 (1:1,000; cat. no. ab80590; Abcam), PI3K (1:1,000; cat. no. ab32089; Abcam), p-PI3K p85 (1:1,000; cat. no. ab278545; Abcam), AKT (1:1,000; cat. no. ab8805; Abcam), p-AKT (1:1,000; cat. no. ab81283; Abcam), Bax (1:1,000; cat. no. ab32503; Abcam), caspase-3 (1:1,000; cat. no. ab13847; Abcam) and GAPDH (1:1,000; cat. no. ab9485; Abcam) primary antibodies in the absence of light. Then cells were incubated with HRP-labeled anti-rabbit IgG secondary antibody (1:4,000; cat. no. ab6721; Abcam) or anti-mouse IgG secondary antibody (1:5,000, cat. no. ab6789, Abcam) for 2 h at room temperature. The bands were imaged with an Pierce™ ECL Western Blotting Substrate kit (cat. no. 32109; Thermo Fisher Scientific, Inc.) and band density was quantified using Quantity One v.6.2 software (Bio-Rad Laboratories, Inc.).
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!