PI 3-kinase p85α Antibody (B-9)

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	Dilute primary Ab at 1:200 and incubate at 4°C overnight

Protocol tips
Dilute primary Ab at 1:200 and incubate at 4°C overnight

Publication protocol

Following 48 h of transfection, total protein from each group was extracted using NP-40 lysate (Beyotime Institute of Biotechnology, Haimen, China) with 1% phenyl-methanesulfonyl fluoride (Beyotime Institute of Biotechnology). The protein concentration of each sample was determined using a Bicinchoninic Acid Protein Assay kit (cat no. P0012S; Beyotime Institute of Biotechnology). A total of 40 µg protein from each group was separated by SDS-PAGE (5% for the spacer gel and 8% for the separating gel) and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk overnight and incubated at 4°C overnight with the following primary antibodies: Rabbit polyclonal anti-APP695 (1:500; cat no. BA0581; Wuhan Boster Biological Technology, Ltd., Wuhan, China), rabbit polyclonal anti-caspase-3 (1:1,000; cat no. bs-0081R; BIOSS, Beijing, China), rabbit polyclonal anti-light chain 3 (LC3; 1:1,000; cat no. 4108; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-autophagy protein 5 (ATG5; 1:200; cat no. BA3525-2; Wuhan Boster Biological Technology, Ltd.), rabbit polyclonal immunoglobulin (Ig)G to phosphorylated (p)-Akt (1:200; cat no. sc-135650; Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit polyclonal IgG to B-cell lymphoma 2 (Bcl-2; 1:200; cat no. sc-492; Santa Cruz Biotechnology, Inc.); mouse monoclonal IgG1 to phosphoinositide 3-kinase p85α (PI3K; 1:200; cat no. sc-1637; Santa Cruz Biotechnology, Inc.), rabbit polyclonal IgG to p-mammalian target of rapamycin (mTOR; 1:100; cat no. sc-8319; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Beclin-1 (1:5,000; cat no. ab55878; Abcam, Cambridge, MA, USA) and rabbit monoclonal to p-mitogen-activated protein kinase 8 (JNK1; 1:100; cat no. EPR17557; Abcam). Membranes were then washed four times for 5 min with Tris-buffered saline containing Tween 20, followed by incubation with the corresponding secondary antibodies, goat anti-rabbit immunoglobulin (Ig)G-HRP and goat anti-mouse IgG-HRP (1:5,000; cat. nos. A0208 and A0216, respectively; Beyotime Institute of Biotechnology), for 90 min at room temperature. For visualization, the membranes were treated with an enhanced chemiluminescence solution (ECL, Qihai Biotec, Shanghai, China), exposed to X-ray film and visualized by autoradiography. The optical density (OD) values of the target protein bands were analyzed using Gel-Pro-Analyzer software 4.0 (Media Cybernetics, Rockville, MD, USA) and normalized to β-actin (1:5,000; HRP-conjugated monoclonal mouse anti-β-actin; cat. no. KC-5A08; KangChen Bio-tech Inc., Shanghai, China).

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Download the product protocol from Santa Cruz Biotechnology for PI 3-kinase p85α Antibody (B-9) below.

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