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 tsc2
Manufactured by Cell Signaling Technology
Sourced in United States
P-TSC2 is a laboratory product offered by Cell Signaling Technology. It is an antibody that detects the phosphorylated form of the TSC2 protein. TSC2 is a key regulator of the mTOR signaling pathway, which plays a role in cellular growth and proliferation. The P-TSC2 antibody can be used to analyze the activation state of the TSC2 protein in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (5 mentions)
P mtor, by Cell Signaling Technology (4 mentions)
P raptor, by Cell Signaling Technology (3 mentions)
P p70s6k, by Cell Signaling Technology (3 mentions)
P ulk1, by Cell Signaling Technology (3 mentions)
P s6k, by Cell Signaling Technology (3 mentions)
4e bp1, by Cell Signaling Technology (3 mentions)
P 4ebp1, by Cell Signaling Technology (3 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
P70s6k, by Cell Signaling Technology (2 mentions)
Tom20, by Santa Cruz Biotechnology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Raptor, by Cell Signaling Technology (2 mentions)
Uqcrc1, by Abcam (1 mentions)
Ampkα1, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Chloroquine, by Merck Group (1 mentions)
P ampk, by Santa Cruz Biotechnology (1 mentions)
15 protocols using p tsc2
1
Western Blotting Analysis of Frozen Tissues
2
Autophagy and STAT3 Regulation Mechanisms
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Materials purchased include the following: Foetal bovine serum (Atlanta Biologicals); Enhanced chemiluminescence (ECL) Western blot detection system (Perkin Elmer, Inc.); Protease/Phosphatase Inhibitor Cocktail, cleaved active caspase‐3 (Asp 175); LC3‐I/LC3‐II; SQSTM1/p62, p‐Akt Ser473, HDAC‐6, phospho‐S6 Ribosomal protein Ser235/236, p‐STAT3 Y705, p‐STAT3 Ser727, Acetyl‐STAT3 Lys685, total‐STAT3, p‐AMPKα Thr172, Total‐ΑΜPΚα, p‐ULK1 Ser555, p‐ULK1 Ser638, Total‐ULK1, p‐TSC2 Ser1387, p‐TSC2 Ser1462, Total‐Tuberin/TSC2, Beclin‐1, BNIP3 and Cathepsin‐D antibodies (Cell Signalling Technology, Inc.); Alexa‐Fluor 488 conjugated, Alexa‐Fluor 647, and Cy3 conjugated secondary antibodies (Molecular Probes); Anti‐trimethyl STAT3 Lys180, and Bafilomycin A1, (EMD Biosciences/Millipore Corp.); ULK1 Inhibitor (MRT68921) (MedChemExpress). The ULK1 siRNA and transfection reagent were obtained from Santa Cruz Biotechnology. The cell death detection ELISA kit was purchased from Roche (Millipore Sigma). All chemicals were of the highest purity commercially available.
3
Western Blot Analysis of Cellular Proteins
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Whole-cell extracts of cultured human cells were prepared in 1.5% n-dodecylmaltoside (Carl Roth, CN26.2) in PBS (Sigma-Aldrich, P44177) as described previously [79 ]. Briefly, gels were loaded with 50 µg of total protein per well, proteins were separated in 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Life Technologies, GE10600023). The following antibodies were used for immunoblotting: UQCRC1 (Abcam, ab110252), GAPDH (Sigma-Aldrich, G9545), HPRT (Abcam, ab10479), LAMP1 (Developmental Hybridoma Bank, H4A3) for mouse; (Abcam, ab24170) samples) for human samples, LC3B (Cell Signaling Technology, 3868), AMPKα1 (Cell Signaling Technology, 2795), p-PRKAA1/AMPKα1 (T172) (Cell Signaling Technology, 2535), FLCN (Cell Signaling Technology, 3697), FNIP1 (Abcam, ab134969), TSC2 (Cell Signaling Technology, 4308), p-TSC2 (S1387) (Cell Signaling Technology, 5584), PIKFYVE (Proteintech, 13361–1-AP), ACAC/ACC (Cell Signaling Technology, 3676), p-ACAC/ACC (Ser79) (Cell Signaling Technology, 3661), RPS6/S6 (Cell Signaling Technology, 2217S), p-RPS6/S6 (Cell Signaling Technology, 4858S), MCOLN1 (Novus, NBP1-92152). Band density quantification was determined using ImageJ after subtraction of background, and normalized against loading controls (HPRT, GAPDH, RPL7).
4
Autophagy Regulation in Pancreatic Cancer
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Human pancreatic cancer cell lines BxPc-3, SW1990, Panc-1 and MiaPaCa-2 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences. They were cultured in 10 % FBS DMEM/RPMI1640 at 37 °C and 5 % CO2. Autophagy inhibitors, 3-MA and chloroquine were purchased from Sigma (San Diego, CA). ASPP2 antibody (sc-53861) is mouse monoclonal IgG1 against amino acids 691–1128 of ASPP2 of human origin. The following antibodies were used for Western blot: AMPK, p-AMPK, Actin (Santa Cruz), p-TSC2, TSC2, Atg5, Atg7, Beclin1, p62, LC3 (Cell signal technology).
5
Western Blot Analysis of Signaling Pathways
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Cells were routinely digested and seeded onto six-well plates at a density of 2 × 105 cells/well. Samples were harvested at the indicated time points followed by the addition of a lysis buffer. The protein concentration was quantified by BCA (Thermo Scientific, Waltham, MA, USA). The same amount of protein was separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with 5% BSA solution, the PVDF membrane was incubated overnight at 4°C with the following primary antibodies: p-AKT, p-mTOR, p-S6k, TSC1, p-TSC2, iNOS, and ARG1 (purchased from Cell signaling Technology, Danvers, MA, USA, ABCAm, Cambridge, UK, and Sigma, USA, respectively). The membrane was then washed three times with TBST and incubated with secondary antibody-linked HRP. Densitometric quantification was carried out using chemiluminescence detection reagents with β-actin (Sigma, USA) as a loading control for whole-cell lysate experiments. The gray values were analyzed by ImageJ software, and all images were normalized.
6
Cell Signaling Pathway Analysis Protocol
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Abemaciclib, BYL719, buparlisib, AZD8186, ipatasertib, AZD5363, and MK2206 were purchased from Selleckchem (Houston, TX, USA). Antibodies against the following proteins were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): p107 (sc-250), p130 (sc-9963), cyclin A (sc-751), cyclin D1 (sc-753), cyclin E (sc-481), CDK2 (sc-163), and GAPDH (sc-47724). Antibodies against Rb (cs#9309), p-Rb (cs#8180), E2F (cs#3742), p16 (cs#92803), p-AKT S473 (cs#4058), p-AKT T308 (cs#9275), AKT (cs#4685), p-TSC2 (cs#3617), TSC2 (cs#4308), and vinculin (cs#13901) were purchased from Cell Signaling Technology (Danvers, MA, USA). Recombinant protein human epithelial growth factor (rhEGF) and recombinant protein human fibroblast growth factor (rhEGF) were purchased from R&D Systems (Minneapolis, MN, USA). Mitomycin C (MMC), propidium iodide (PI), RNase, and sodium dodecyl sulfate (SDS) were purchased from Sigma Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco’s modified Eagle’s medium (DMEM) medium were purchased from Welgene (Daejeon, Korea). All chemicals and reagents were of analytical grade and were obtained from commercial sources.
7
Comprehensive Protein Extraction and Analysis
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Protein extraction was performed by lysing the cells in RIPA (radio-immunoprecipitation assay) buffer. The protein concentration was determined using the DC protein assay, and equal amounts of protein were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked with 5% nonfat milk in TBST for 1 h. The primary antibodies were added and incubated overnight at 4 °C with shaking. The membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. The primary antibodies of TTl1, FGFR3, α-actin, p53, p21, ATF3, FAS, TOM20, mtTFA, and PGC1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); TELO2, Cyclin D1, and γ-H2A.x were obtained from Abcam (Cambridge, UK); and p-mTOR, mTOR, p-ATM, ATM, p-p53 (ser15), MDM2, DEC1, p-c-JUN, c-JUN, p-Akt, Akt, p-p38, p38, p-PI3K, PI3K, p-ERK, ERK, p-Chk2, Chk2, p-AMPK, AMPK, p-eIF2α eIF2α, p-P70S6K, P70S6K, p-TSC2, TSC2, and 4EBP1 were obtained from Cell Signaling (Danvers, MA, USA).
8
Western Blot Analysis of Cellular Signaling
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T cells were lysed in Ripa buffer containing a protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 4 °C at 12,000× g for 10 min, separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 and incubated with various antibodies recognizing pAMPKα1 (T172), pAMPKα1 (S485), pS6 (S235/236), peIF4E (S209), pULK1 (S555), ATG7, AQP9, PGC1α, TFAM, OPA1, pDRP1 (S616), ID2, ID3, cMyb, cMyc, Blimp1, T-bet, ZEB2, FOXO1, TCF1, TRAF6, pTSC2 (S1387), Eomes, HIF-1α and β-actin (Cell Signaling Technology) and the Complex I subunit NDUFA9 (Abcam, Cambridge, MA, USA). The membranes were imaged using a BioRad Chemidoc MP (Bio-Rad, Hercules, CA, USA) after a second incubation step with a horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Cell Signaling) secondary antibody [18 (link)].
9
Intestinal Mucosal Protein Quantification
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The Western blot was used to detect liver kinase B1 (LKB1), ras homolog enriched in the brain (Rheb), tuberous sclerosis complex 2 (TSC2), Raptor, mTOR, S6K1, and 4EBP1 phosphorylation and expression levels of total proteins in jejunal and ileal mucosa, respectively. The jejunal and ileal mucosa samples were homogenized, and protein concentrations were measured using the bicinchoninic acid assay method with BSA as standard (Catalog# P0010, Beyotime Institute of Biotechnology, Shanghai, China). The following first antibodies were used for protein quantification: LKB1 (1:1,000, Cell Signaling Technology); Raptor (1:1,000, Cell Signaling Technology); Rheb (1:1,000, Cell Signaling Technology); mTOR (1:1,000, Cell Signaling Technology); TSC2 (1:1,000, Cell Signaling Technology); S6K1 (1:1,000, Cell Signaling Technology); 4EBP1 (1:1,000, Cell Signaling Technology); p-mTOR (1:1,000, Cell Signaling Technology); p-TSC2 (1:1,000, Cell Signaling Technology); p-S6K1 (1:1,000, Cell Signaling Technology); and p-4EBP1 (1:1,000, Cell Signaling Technology) and p-4EBP1(1:1,000, Cell Signaling Technology).
10
Sunitinib, autophagy, and apoptosis signaling
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Sunitinib, CQ and all fluorescent dyes were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The primary antibodies against Bcl-2 (sc-492), Bax (sc-70407) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Primary antibodies against Beclin-1 (ab62557), autophagy related 5/12 (Atg5/12; ab78073) were purchased from Abcam (Cambridge, MA, USA). Primary antibodies against light chain 3 (LC3) I/II (no. 4108), p53 (no. 9282), caspase-3 (no. 9662), caspase-9 (no. 9508), phosphorylation of histone H3 (no. 9711), phosphoinositide 3-kinase (PI3K; no. 4255), Akt (no. 4685), phosphorylated (p)-Akt (no. 4058), tuberous sclerosis complex 2 (TSC2; no. 4308), p-TSC2 (no. 3617), p-mechanistic target of rapamycin (p-mTOR; no. 5536) and mTOR (no. 4517), p70 ribosomal S6 kinase (p70S6K; no. 2708) and p-p70S6K (no. 9204) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
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