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 mtor
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-mTOR is a primary antibody that specifically targets the mammalian target of rapamycin (mTOR) protein. mTOR is a serine/threonine protein kinase that plays a central role in the regulation of cell growth, proliferation, and metabolism. Anti-mTOR is a tool used in research applications to detect and study the mTOR protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti p mtor, by Abcam (28 mentions)
Anti akt, by Abcam (23 mentions)
Anti p akt, by Abcam (19 mentions)
Anti gapdh, by Abcam (16 mentions)
Anti bax, by Abcam (11 mentions)
Anti bcl 2, by Abcam (10 mentions)
Anti pi3k, by Abcam (8 mentions)
Anti p62, by Abcam (8 mentions)
Anti beclin1, by Abcam (7 mentions)
Anti p pi3k, by Abcam (6 mentions)
Anti p70s6k, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Anti cleaved caspase 3, by Abcam (5 mentions)
Anti lc3b, by Abcam (4 mentions)
Anti n cadherin, by Abcam (4 mentions)
Anti 4e bp1, by Abcam (4 mentions)
Anti atg5, by Abcam (4 mentions)
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Abcam (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
65 protocols using anti mtor
1
Protein Expression Analysis in SKOV3 Cells
2
Western Blot Analysis of Autophagy and Apoptosis Markers
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Samples were lysed in a lysis buffer containing the following: 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulphonyl fluoride (PMSF), with protease inhibitor cocktail (pepstatin 1 g/mL, aprotinin 1 g/mL, leupeptin 1 g/mL). Protein concentrations were measured using the BCA method and 40 μg of proteins were loaded and separated using SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and blocked in 5% nonfat milk or 5% bovine serum albumin. Membranes were incubated at 4 ? overnight with the following antibodies: anti-SOD1 (1:2000, Abcam), anti-LC3 (1:1000, Sigma), anti-p62 (1:1000, CST), anti-Beclin1 (1:1000, CST), anti-mTOR (1:1000, Abcam), anti-ATG5 (1:500, MBL), anti-p62 (1:500, MBL), anti-calpain 1 (1:1000, Abcam), anti-Bip (1:1000, CST), IRE1α (1:500, CST), CHOP (1:1000, CST) and PDI (1:1000, CST), anti-cleaved-caspase-12 (1:500, CST) and anti-β-actin (1:10000, Sigma). The membranes were then incubated with appropriate peroxidase-conjugated secondary antibodies for 2 hours. Protein bands were visualized using ECL (Pierce, USA) and an image analyzer was used to quantify the densities of interested protein bands (Bio-Rad, Image lab 4.1).
3
Protein Expression Analysis via Western Blot
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Cells were disrupted by lysis buffer and protein content was determined using the BCA Protein Assay Kit. The quantified protein samples were then separated by SDS-PAGE and transferred to PVDF membranes, followed by sealing with 5% skim milk for 1 hour at room temperature. The PVDF membranes carrying the samples were incubated with primary antibodies overnight at 4°C, and then with secondary antibody for 2 h at room temperature. The signal was displayed with an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.). And, the protein expression levels were semi-quantified using Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) software. The antibodies used in this study were purchased from Abcam and were used at the following concentrations: anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-cleaved caspase3 (1:100), anti-caspase3 (1:500), anti-PDE3B (1:2000), anti-p-AMPK (1:1000), anti-AMPK (1:1000), anti-p-mTOR (1:1000), anti-mTOR (1:10,000), anti-GAPDH (1:2500), and goat anti-rabbit IgG H&L (HRP) (1:2000).
4
Gastric Cancer Protein Expression Analysis
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After 48 hr’s transfection, the proteins of gastric cancer cells were extracted using RIPA buffer containing 1% protease inhibitors (Roche, Basel, Switzerland). We used the BCA assay to determine protein concentration. The proteins were separated using SDS–PAGE at 20 μg/lane and transferred them to a nitrocellulose membrane (Millipore, Bedford, MA). Then we blocked the membrane with 5% non-fat milk for 2 hrs, incubated them with primary antibodies at 4 °C for one night, and the second antibodies for 1 hr at room temperature. After washing with TBST 3 times, we detected the protein bands by ECL. The primary antibodies to anti-NKAP, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, anti-P70S6K, and anti-GAPDH were purchased Abcam. We utilized Image J to quantify the density of each band.
5
Western Blot Analysis of mTOR Pathway Proteins
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Lysates were prepared in 1x Dulbecco’s Phosphate Buffer Saline (DPBS—Invitrogen) containing 1% Nonidet P 40 substitue (NP40 –Signa-Aldrish, MO, USA) and protease inhibitor cocktail (Roche Diagnostics, IN, USA). Total protein was determined by Lowry’s method and 25 mg was loaded on a 4–12% gradient SDS–polyacrylamide gel electrophoresis (Invitrogen). Proteins were transferred to 2 μM nitrocellulose membrane using the Trans-blot turbot kit (Bio-Rad) and blotted over-night with anti-mTOR (rabbit polyclonal, abcam), anti-Rictor (rabbit polyclonal, Cell Signaling), anti-Raptor (rabbit polyclonal, Cell Signaling), anti-Atg5 (mouse monoclonal, Cell Signaling), anti-Atg7 (mouse monoclonal, Cell Signaling), anti-pS6K1 (rabbit polyclonal, Abcam), anti-S6K1 (rabbit polyclonal, Abcam), anti-peIF4E (Ser209) (rabbit polyclonal, Cell Signaling), anti-eIF4E (rabbit polyclonal, Cell Signaling), anti-E1/E2 (rabbit polyclonal, gift from Olivier Schwartz laboratory), anti-C (monoclonal antibody, gift from Olivier Schwartz laboratory), anti-GFP (rabbit polyclonal, Cell Signaling) or anti-GAPDH (rabbit polyclonal, Cell Signaling). Secondary HRP-coupled Abs was detected using ECL Plus (Amersham Pharmacia Biotech).
6
Western Blot Analysis of Liver Protein Markers
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Protein concentrations (n = 6) were determined in the supernatant of liver tissues by classic BCA protein assay (Beyotime). Equal protein of each sample was fractionated onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane by a Bio-Rad Western blot apparatus. The membranes were blocked with 5% fat-free milk or 5% bovine serum albumin and then probed with the following primary antibodies for 12 hours at 4°C: GAPDH (1 : 2000), Anti-Bax (1 : 2000), Anti-Bcl-2 (1 : 1000), Anti-Rheb (1 : 2000), Anti-Tuberin (1 : 1000), Anti-p-Tuberin (1 : 500), Anti-mTOR (1 : 800), Anti-p-mTOR (1 : 800), Anti-Notch1 (1 : 1000), Anti-Cyclin D (1 : 1000), Anti-p70S6K (1 : 1000), and Anti-4E-EIF (1 : 1000) (Abcam, Cambridge, UK). The membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 2000~1 : 3000, Abcam, Cambridge, UK) and visualized with an enhanced chemiluminescence (ECL) detection kit (Millipore). Bands were quantified using quantity one 4.40 software (Bio-Rad, CA, USA).
7
Western Blot Analysis of PI3K/AKT/mTOR Pathway
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All the patients signed the written informed consents before enrollment, and the procedures were approved by the ethical committees of The First Affiliated Hospital of Shandong First Medical University (No. 2017-S007). And 3 cases of ccRCC tissues (including cancer and adjacent non-cancerous), which were collected from 3 patients (2 males and 1 females, age from 54 to 65, Fuhrman nuclear grading with 1 G2 + 2 G3, TNM staging with 2 T1 + 1 T2) (Edge & Compton, 2010 (link)), were used for WB analysis as described previously (Zhao et al., 2017 (link)). After incubated with the corresponding primary antibodies: anti-PI3K (1:1,000, rabbit, #4249; Cell signaling Technology, Danvers, MA), anti-AKT (1:1,000, rabbit, #4691; Cell signaling Technology, Danvers, MA), anti-mTOR (1:2,000, rabbit, ab2732; Abcam, Cambridge, MA), and anti-β-Actin (1:1,000, mouse, BM0627; BOSTER, Beijing, China), horseradish peroxidase (HRP) conjugated secondary antibodies (1:5,000, rabbit, SA00001-2 & mouse, SA00001-1; Proteintech, Wuhan, China) were used to detect the proteins. Then the blots were visualized by enhanced chemiluminescence (Amersham Imager 600, GE Healthcare, Marlborough, MA). Protein ladder (MAN0011772, Thermo Scintific, Waltham, MA) was used to label the corresponding proteins, and Image J software was used for quantification.
8
Western Blot Analysis of Protein Targets
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Western blot was performed as previously reported13 . Briefly, whole-cell lysates were prepared and protein concentration was estimated using a BCA Protein Assay kit (Pierce, Rockford, MA, USA). The immune-blots were probed with primary antibodies overnight at 4 °C followed by secondary antibodies for 1 hr. The following primary antibodies were used according to the manufacturer’s protocols: rabbit anti-DDX5, anti-mTOR, anti-p-mTOR (phospho S2448), anti-S6K1, anti-p-S6K1 (phospho T389), mouse anti-β-actin, peroxidase conjugated goat anti-rabbit or anti-mouse IgG (all from Abcam). The blots were visualized using enhanced chemiluminescence detection kit (Thermo). The bands were scanned and quantified by densitometric analysis using Image J software (National Institutes of Health, Bethesda, MD, USA).
9
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.).
10
Investigating Molecular Pathways in Osteoarthritis
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The antibodies used in this study were as follows: anti-P2X7 (Abcam, Cambridge, UK; cat. no. ab109054), anti-collagen II (Abcam; cat. no. ab34712), anti-MMP13 (Abcam; cat. no. ab39012), anti-AMPKα1 (Abcam; cat. no. ab32047), anti-mTOR (Abcam; cat. no. ab109268), anti-NLRP3 (Proteintech; cat. no. 19771-1-AP), anti-caspase-1 (Proteintech; cat. no. 22915-1-AP), anti-LC3B (Abcam; cat. no. ab192890), anti-Beclin-1 (Abcam; cat. no. ab62557), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech; cat. no. 10494-1-AP), and horseradish peroxidase (HRP)-labeled IgG (Beyotime; cat. no. A0208). The reagents used in the experiment were as follows: MIA (Sigma, St. Louis, MO, USA; cat. no. I2512), the P2X7 receptor agonist BzATP (Sigma; cat. no. B6396), the mTOR activator MHY1485 (Sigma; cat. no. SML0810), the mTOR inhibitor rapamycin (Sigma; cat. no. V900930), the NLRP3 inhibitor CY-09 (Sigma; cat. no. SML2465), the AMPK activator A-769662 (Sigma; cat. no. SML2578), and the AMPK inhibitor compound C (Sigma; cat. no. P5499). The concentrations, dosages, and preparation of the reagents were described previously [43 (link), 45 (link)].
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