Western blotting was performed on snap-frozen tissue samples (SED: n = 3; EX: n = 3; EX + DOX: n = 3). Tissue lysates were prepared by homogenization and the protein concentration was assessed as previously described (Kolwicz et al. 2009 (link)). Equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membrane (BioRad, Hercules, CA). Primary antibodies for: phosphorylated (p)-AMPK at threonine(thr)172, AMPK, p-Akt at serine (ser) 473, Akt, p-TSC2(ser1387), p-TSC2(thr1462), TSC2, p-mTOR(ser2481), p-mTOR(ser2448), mTOR, p-Raptor(ser792), Raptor, p-p70S6K(thr387), p70S6K, p-ULK1(ser317), p-ULK1(ser757),and ULK1 (Cell Signaling, Danvers, MA) were used. Signals were visualized by enhanced chemiluminescence, digitized, and quantified with Image J software (NIH, Bethesda, MD).
P mtor
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany
P-mTOR is a phosphorylated form of the mammalian target of rapamycin (mTOR) protein. mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. The phosphorylation of mTOR is an important regulatory mechanism for its biological functions.
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Lab products found in correlation
P akt, by Cell Signaling Technology (303 mentions)
β actin, by Cell Signaling Technology (88 mentions)
Gapdh, by Cell Signaling Technology (85 mentions)
P p70s6k, by Cell Signaling Technology (82 mentions)
P70s6k, by Cell Signaling Technology (73 mentions)
P 4ebp1, by Cell Signaling Technology (73 mentions)
P pi3k, by Cell Signaling Technology (70 mentions)
Beclin 1, by Cell Signaling Technology (64 mentions)
Bcl 2, by Cell Signaling Technology (62 mentions)
P erk, by Cell Signaling Technology (57 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (55 mentions)
P ampk, by Cell Signaling Technology (51 mentions)
Cleaved caspase 3, by Cell Signaling Technology (50 mentions)
Pvdf membrane, by Merck Group (48 mentions)
β actin, by Santa Cruz Biotechnology (43 mentions)
4e bp1, by Cell Signaling Technology (41 mentions)
P erk1 2, by Cell Signaling Technology (39 mentions)
Caspase 3, by Cell Signaling Technology (38 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Erk1 2, by Cell Signaling Technology (28 mentions)
619 protocols using p mtor
1
Western Blotting Analysis of Frozen Tissues
2
Characterization of Signaling Pathways in Human Myeloma
3
Western Blot Analysis of Cellular Signaling
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Cells were rinsed with ice-cold PBS 1X and lysed in lysis buffer (15 mM HEPES, 50 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.25% glycerol, 0.5% laurylmaltoside, 5 μM GDP, 1 μM microcystin (Enzo Life Sciences), 1 mM sodium orthovanadate and cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich/Roche). After centrifugation, proteins were quantified using the Bradford assay (Bio-Rad, CA). Thirty micrograms of proteins were separated by SDS–PAGE (Biorad) and transferred to PVDF membranes (Millipore). After blocking, membranes were incubated overnight at 4°C with the following primary antibodies: Bcl-2 (#M0887, DAKO), actin (MAB1501, Merck Millipore), Mcl-1 (#5453), Bcl-xL (#2764), PARP(#9542), caspase-3 (#9662), P-Akt (Thr308; #13038), P-Akt (Ser473; #4060), Akt (#9272), P-4E-BP1 (Thr70; #4370), 4E-BP1 (#9644), P-p70S6K (Thr389; #9205) and p70S6K (#9202), P-mTOR (Ser2448; #5536), P-mTOR (Ser2481; #2974), p-CamKII (Thr286; #12716), p-AMPK (Ser485; #4184), AMPK (#2532) (Cell Signaling Technology). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). Revelation was done using Clarity Western ECL (Biorad). Western blots shown are from one experiment representative of at least three independent experiments and cell lysates. Signals were quantified by pixel densitometry using the ImageJ software.
4
Osteosarcoma Cell Lines: Culture and Drug Treatments
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Human osteosarcoma cell lines MG-63, Saos-2 and U-2 OS were purchased from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China) and cultured in McCoy’s 5a medium (Gibco, Los Angeles, CA) containing 10% fetal bovine serum (Gibco) and ampicillin and streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. All cell lines were used within 20 passages. The antibody against HSP90AA1was obtained from Proteintech. The antibodies against LC3, p62, cleaved PARP, Akt, p-Akt, mTOR, p-mTOR, JNK, p-JNK, p38, p-p38 and actin were obtained from Cell Signaling Technology. Cisplatin, doxorubicin, methotrexate, bafilomycin A1, rapamycin, LY294002 and 3-methyladenine were purchased from Sigma Aldrich (St. Louis, MO, USA).
5
Western Blot Analysis of U87 Cells
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U87 cells were washed with ice-cold PBS before lysis in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Cells were scraped off from six-well plates and agitated at 4°C for 10 min. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were mixed at 1∶1 ratio with 2× Laemmli buffer. Protein lysates were heated at 95°C for 5 min, separated by 10% Mini-PROTEAN gels (Bio-Rad), transferred onto 0.45 µm nitrocellulose membranes, and probed with antibodies against p-STAT1 (Y701), total STAT1, NF-κB p65, p-Akt, total Akt, p-mTOR, total mTOR, PKR (Cell Signaling), VSV, GFP, NF-YA, NF-YB, NF-YC, p-p70 S6 kinase, total p70 S6 kinase, PI3K, p21, Rb, caspase-3, caspase-9, XIAP, survivin, β-tubulin (Santa Cruz), acetylated H3 (Millipore), and total histone H3 (Abcam). Secondary incubation was performed with anti-goat, anti-rabbit, or anti-mouse IgG HRP-linked antibodies (Cell Signaling). Proteins were visualized using HyGlo chemiluminescent HRP antibody detection reagent (Denville Scientific Inc.) and a Fuji LAS-3000 cooled CCD camera (FUJIFILM).
6
Autophagy and Apoptosis Pathway Analysis
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BCa cell lines were seeded in six-well plates, and after 24 h of culture, the cells were treated with NA at different dose and time. Western blotting was performed using specific antibodies against, Atg3, Atg5, Atg7, Atg12, beclin 1, LC3A, LC3B (Autophagy antibody sampler kit #4445, Cell Signaling), mTOR (#2972), pmTOR (#2974), p70S6K (#2708), phospho-p70S6K (#9234), BAX (#2772), BCL-2 (#2876), cleaved caspase-3 (#9661), cleaved caspase-9 (#9505), PARP (#9542) and cleaved PARP (#9541) (Cell Signaling), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Protein–antibody complexes were detected with the enhanced chemiluminescence method as described earlier (Suman et al, 2013b (link)).
7
In vivo Protein Analysis after SCI
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For in vivo protein analysis, a spinal cord segment at the lesion epicentre was dissected at 7 and 14 days and immediately stored at −80°C. Proteins from animal tissue or PC12 cells were first quantified by the BCA reagent method. We separated the proteins on 8% (w/v) or 12% (w/v) gels and transferred the proteins onto polyvinylidene fluoride membrane (Bio‐Rad, Hercules, CA, USA). Membranes were blocked with 5% (w/v) milk (Bio‐Rad) in TBS with 0.05% Tween 20 (TBST) for 2 hours at room temperature and then were incubated overnight at 4°C with primary antibodies of FGF1 (1:1000; Abcam), ace‐tubulin (1:2000; Cell Signaling Technology), Tau (1:1000; Abcam), GAP43 (1:1000; Abcam), MBP (1:1000; Abcam), p‐mTOR (1:1000; Cell Signaling Technology), mTOR (1:1000; Cell Signaling Technology), ATG7 (1:1000; Bio‐world), Beclin1 (1:1000; Abcam), LC‐3 I/II (1:1000; Cell Signaling Technology), PRDX1 (1:1000; Cell Signaling Technology) and GAPDH (1:10000; Bio‐world). Next, the membranes were washed with TBST 3 times and treated with horseradish peroxidase‐conjugated secondary antibodies for 60 minutes. All signals were detected by ChemiDocXRS + Imaging System (Bio‐Rad). All experiments were repeated 3 times for accuracy.
8
Evaluating SNX-2112 Cytotoxicity and Signaling
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SNX-2112 was synthesized as previously described in our lab with >98.0% purity [42 (link)], dissolved in Dimethyl sulfoxide (DMSO) to obtain a 100 mM stock solution, and stored at −20°C. TRAIL was purchased from Merck Millipore (Waltham, MA, USA). N-Acetyl-cysteine (NAC) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Bafilomycin A1 (BFA) was purchased from Selleck Chemicals (Shanghai, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies for GAPDH, Bcl-2, Bcl-xL, FLIP, pro-caspase 3, cleaved-caspase 3 (c-caspase 3), cleaved-caspase 8 (c-caspase 8), cleaved-PARP (c-PARP), Akt, p-Akt (Ser473), DR4, DR5, LC3, Beclin1, Atg7, p62, p-mTOR, p-S6, p-4EBP1, p53, p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (CST; Beverly, MA, USA).
9
Western Blot Protocol for Protein Analysis
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The Western blot analysis was performed as reported previously14 (link). Total cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, and incubated with the primary antibodies listed next for 1 h with shaking. The primary antibodies against TG2, β-catenin, p-β-catenin, non-p-β-catenin, Ampka, p-Ampka, Foxo3a, p-Foxo3a, p65, p-p65, mTOR, p-mTOR, Axin2, Tcf1, Sirt1, and IκB were purchased from Cell Signaling Technology (Danvers, MA); antibodies against Sox9, Runx2, Mmp3, Mmp13, and Adamts5 were from Abcam (Cambridge, UK); the antibodies against LaminB and haemagglutinin (HA) were from Santa Cruz Biotechnology (Dallas, TX), and β-actin antibody was from Sigma Aldrich (St. Louis, MO). Membranes were washed, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. The membrane signal was visualized using Supersignal Chemiluminescent Substrates (Thermo Fisher Scientific, Waltham, MA).
10
Protein Extraction and Western Blotting
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