To examine the expression of the proteins in both cell lines, SKOV3-CDDP and SKOV3 cells were seeded at 2 × 105 cells per well in 6-well plates and cultured overnight. The two cell lines were treated with PBS, miR497, miR497-HENPs, TP, TP-HENPs and miR497/TP-HENPs for 48 h. Then, 150 µl RIPA buffer (Sigma–Aldrich) was added and lysed on ice for 5 min. The lysate was collected and centrifuged at 12,000 g for 15 min at 4 °C. The protein content in the supernatant was quantified by a BCA protein assay kit. Protein samples were separated by SDS–PAGE and transferred to PVDF membranes, and the membranes were blocked with 5% blocking buffer for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-calnexin, anti-TSG101, anti-CD9, anti-CD47, anti-GAPDH, anti-PI3K, anti-p-PI3K and anti-mTOR (purchased from Abcam) and anti-p-mTOR, anti-AKT, anti-p-AKT and HIF-α (obtained from CST). The membranes were washed three times, and then at room temperature, the secondary antibody was incubated for 1 h. All strips were visualized using a Bio–Rad Imaging System (Bio–Rad, USA).
Anti p pi3k
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-p-PI3K is a primary antibody that recognizes the phosphorylated form of the p85 subunit of phosphoinositide 3-kinase (PI3K). PI3K is an important signaling enzyme involved in various cellular processes. This antibody can be used to detect the activated state of PI3K in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti pi3k, by Abcam (26 mentions)
Anti p akt, by Abcam (23 mentions)
Anti akt, by Abcam (20 mentions)
Anti gapdh, by Abcam (11 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Anti mtor, by Abcam (6 mentions)
Anti bax, by Abcam (5 mentions)
Anti cleaved caspase 3, by Abcam (4 mentions)
Anti p mtor, by Abcam (4 mentions)
Anti bcl 2, by Abcam (4 mentions)
Anti e cadherin, by Abcam (4 mentions)
Anti tsg101, by Abcam (3 mentions)
Anti p mtor, by Cell Signaling Technology (3 mentions)
Anti β actin, by Abcam (3 mentions)
Anti cyclin d1, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Anti calnexin, by Abcam (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
36 protocols using anti p pi3k
1
Protein Expression Analysis in SKOV3 Cells
2
Western Blot Analysis of Protein Signaling
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Total protein extracts were resolved on a 10% SDS-PAGE and transferred onto a Hybond PVDF membrane (GE Healthcare, Little Chalfont, UK). Then, the membranes were probed with primary antibodies, followed by the incubation with horseradish peroxidase-conjugated secondary antibody (Abcam; dilution 1 : 10 000). The protein bands were quantified with the enhanced chemiluminescence solution (ECL, GE Healthcare) with an ImageJ software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies were used: anti-MMP-9 (Abcam; dilution 1 : 1000), anti-MMP-2 (Abcam; dilution 1 : 2000), anti-Bcl-2 (Cell Signaling Technology, Danvers, MA, USA; dilution 1 : 1000), anti-cleaved caspase-3 (Cell Signaling Technology; dilution 1 : 1000), anti-Bax (Abcam; dilution 1 : 5000), anti-PI3K (Abcam; dilution 1 : 1000), anti-mTOR (Cell Signaling Technology; dilution 1 : 1000), anti-AKT (Abcam; dilution 1 : 10 000), anti-p-PI3K (Abcam; dilution 1 : 1000), anti-p-mTOR (Cell Signaling Technology; dilution 1 : 1000) and anti-p-AKT (Abcam; dilution 1 : 500). β-Actin was used as a protein control.
3
Western Blot Analysis of Apoptosis Regulators
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Thirty μg of protein was separated by 12% SDS-PAGE and transferred onto a 0.22-μm PVDF membrane (Millipore, Boston, MA, USA). The following primary antibodies were used to incubated the membrane at 4°C overnight: anti-Bcl2L12 (1:500, Abcam, Cambridge, UK), anti-EGFR (1:500, Abcam), anti-PI3K (1:400, Cell Signaling Technology (CST), Boston, MA, USA), anti-p-PI3K (1:200, CST), anti-caspase-7 (1:300, Abcam), and anti-β-actin (1:800, Abcam) that was used as the internal reference. After washing and incubating with the corresponding HRP-conjugate secondary antibodies, the membrane was analyzed with the Enhanced Chemiluminescent (ECL) Western blotting Kit (Millipore) in a Gel Imaging System (Bio-Rad).
4
Protein Expression and Regulation Analysis
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Proteins were extracted using NP40 lysis buffer (Beyotime Institute of Biotechnology) and the bicinchoninic acid assay was applied to determine protein concentration. Proteins (20 µg) were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (cat. no. FFP28; Beyotime Institute of Biotechnology). The membrane was blocked using 5% skimmed milk at room temperature for 60 min and was then incubated with the following rabbit anti-human primary antibodies at 4°C for 12 h: Anti-HIF-1α (1:500; cat. no. ab51608; Abcam); anti-survivin (1:5,000; cat. no. ab76424; Abcam); anti-cleaved caspase-3 (1:500; cat. no. ab2302; Abcam); anti-phosphorylated (p)-mTOR (1:1,000; cat. no. ab109268; Abcam); anti-mTOR (1:2,000; cat. no. ab2732; Abcam); anti-p-Akt (1:500; cat. no. ab38449; Abcam); anti-Akt (1:500; cat. no. ab8805; Abcam); anti-p-PI3K (1:1,000; cat. no. ab182651; Abcam); anti-PI3K (1:1,000; cat. no. ab191606; Abcam); and anti-GAPDH (1:2,500; cat. no. ab9485, Abcam). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; cat. no. ab205718; Abcam) at room temperature for 1 h. The bands were visualized by enhanced chemiluminescence (EMD Millipore), and densitometry was performed using the Bio-Rad ChemiDoc system with Image Lab software version 6.0 (Bio-Rad Laboratories, Inc.).
5
Molecular Mechanisms of LYC Treatment
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LYC, complete Freund’s adjuvant (CFA), and corn oil were obtained from Solarbio (Beijing, China). The primary antibodies, including anti-GAPDH, anti-p-PI3K, anti-PI3K, anti-p-Akt, anti-Akt, anti-p-m-TOR, anti-m-TOR, anti-Bax, anti-bcl-2, anti-E-cadherin, anti-N-cadherin antibodies (at 1:1000 dilution respectively), HRP-labelled goat anti-mouse IgGs (at 1:2000 dilution), and antibodies for immunofluorescence staining were purchased from Abcam (Cambridge, MA, USA). Dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA).
6
Western Blot Analysis of Apoptosis and EMT Markers
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Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane. TBST containing 5% skim milk was sealed at 37 °C for 2 h. The membranes were then incubated with primary antibodies including anti-Bcl-2 (#ab32124, 1/1000; Abcam), anti-Bax (#ab32503, 1/1000; Abcam), anti-Vimentin (#ab92547, 1/1000; Abcam), anti-E-cadherin (#ab231303, 1/1000; Abcam), anti-Cyclin D1 (#ab16663, 1/200; Abcam), anti-SOX9 (#ab185230, 1/1000; Abcam), anti-p-PI3k (#ab182651, 1/1000; Abcam), anti-PI3K (ab191606, 1/1000; Abcam), anti-p-AKT (#ab38449, 1/1000; Abcam), anti-AKT (ab131168, 1/1000; Abcam) at 4 °C overnight, the membrane was washed with TBST three times, five min each time. Then, the membranes were incubated with secondary antibodies for 2 h at room temperature, the membranes were washed with TBST three times, five min each time. ECL kit was used for chemiluminescence detection. Image Pro Plus 6.0 was used to analyze the relative expression of target protein, which was expressed by the ratio of density ratio to GAPDH.
7
Protein Expression Analysis by Western Blotting
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The cells were harvested and lysed with IPH buffer and protease-inhibitor, then the proteins were denatured by boiling in SDS buffer and were loaded on 12% SDS-polyacrylamide gels (Bio Rad). Separated proteins were transferred onto 0.2-μM nitrocellulose membranes by turbo blotting for 7 min at 2.5 A and 25 V using the Bio Rad system. Unspecific protein binding was blocked by incubation in 5% non-fat milk in TBS-Tween 0.05% for 1 h at room temperature or overnight at 4°C. Membranes were subsequently incubated with anti-Cyclin D1 (Abcam, USA, 1: 1000), anti-Cyclin D3 (CST, USA, 1: 1000), anti-Bax (CST, USA, 1: 2000), anti-Bcl2 (Abcam, USA, 1: 2000), anti-PI3K (Abcam, USA, 1: 2000), anti-p-PI3K (Abcam, USA, 1: 500), anti-Akt (CST, USA, 1: 2000), anti-p-Akt (CST, USA, 1: 1000), or anti-GAPDH (Abcam, USA, 1: 10 000). Then, membranes were washed in TBS-Tween and incubated with HRP-conjugated secondary antibody. Signals were detected with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and a LAS 4000 imager (GE Healthcare).
8
Extracellular Vesicle Protein Profiling
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Cell or tissue samples were lysed using RIPA reagent supplemented with PMSF (Roche), protease inhibitor (Roche), and phosphatase inhibitor (BestBio). Proteins were separated by SDS‐PAGE and blotted onto PVDF membranes. The primary Abs anti‐ALIX, anti‐TSG101, anti‐CD63, anti‐CD81, anti‐Calnexin, anti‐EphB2, anti‐p‐PI3K, anti‐PI3K, anti‐p‐AKT, anti‐AKT, and anti‐GAPDH were purchased from Abcam. Protein bands could be imaged after development of the blots using the chemiluminescence kit (Vazyme).
9
Exosome Protein Marker Analysis
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The protein samples were extracted and separated by 10% SDS-PAGE gel, and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skimmed milk and incubated overnight, using the following main detection antibodies at 4°C: anti-CD63 (1:1,000; Abcam, UK), anti-TSG101 (1:1,000; Abcam), anti-Alix (1:1,000; Abcam), anti-Hsp90 (1:1,000; Abcam), anti-GRP94 (1:1,000; Abcam), anti-Cytochrome (1:1,000; Abcam), anti-p-pi3k (1:1,000; Abcam), anti-pi3k (1:1,000; Abcam), anti-p-akt (1:1,000; Abcam), anti-akt (1:1,000; Abcam), anti-E-cadherin (1:1,000; Abcam), anti-vimentin (1:1,000; Abcam), or anti-b-actin (1:5,000; Proteintech, USA). We washed 3 times with TBS-T and the membranes were cultured with the secondary antibody at 24°C for 1 h. The western blots were pictured using an ECL Reagent (Pierce, USA) and the density was verified using ImageJ software (NIH, USA).
10
EGCG Modulates PTEN/PI3K/Akt Pathway
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EGCG (purity > 99%) was purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) was provided by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sodium salicylate was purchased from Shanghai Xilong Biochemical Technology Co., Ltd. (Shanghai, China). Total RNA Rapid Extraction Kit, HiFiScript Quick gDNA Removal cDNA kit, and SYBR green PCR Master Mix were obtained from Beijing Biotek Biotechnology Co., Ltd. (Beijing, China). The primers used for quantitative polymerase chain reaction (qPCR) were synthesized by the Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). RIPA buffer and BCA protein quantification kit were ordered at Beyotime Biotechnology (Shanghai, China). The primary antibodies included anticleaved caspase-3 (9664, CST, USA), anti-PTEN (9559, CST, USA), anti-Akt (4691, CST, USA), anti-p-Akt (4060, CST, USA), anti-mTOR (2983, CST, USA), anti-p-mTOR (5536, CST, USA), anti-GAPDH (5174, CST, USA), anti-PI3K (ab227204, Abcam, UK), and anti-p-PI3K (ab182651, Abcam, UK). The Size Separation Master Kit with Split Buffer (12–230 kDa/66–440 kDa) was obtained from ProteinSimple (California, USA)
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