Western blot analysis was carried out as described (Corno et al., 2017 (link)). Briefly, samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk, and then incubated overnight at 4°C with the following antibodies: anti-phospho-Akt (Ser473), anti-Akt (BD Science, Franklin Lakes, NJ, United States), anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), anti-ERK1/2, anti-AR (Millipore, Burlington, MA, United States); anti-Hsp90 (ac-Lys294) (Novus, Centennial, Colorado, United States), anti-Hsp90 (Santa Cruz Biotechnology, Dallas, TX, United States), anti-acetylated alfa-tubulin (Sigma-Aldrich, Milan, Italy), anti-Bax and anti-FLIPL (Sigma-Aldrich, Milan, Italy), anti-p53 (Dako, Santa Clara, CA, United States), anti-cleaved caspase-3 (Asp175) and anti-cleaved caspase-7 (Asp198) (Cell Signaling, Danvers, MA, United States). Anti-vinculin (Sigma-Aldrich, Milan, Italy), anti-β-tubulin (Abcam, Cambridge, United Kingdom) or anti-actin (Sigma) antibodies were used as control for loading. Antibody binding to blots was detected by chemo-luminescence (Amersham Biosciences, Cologno Monzese, Italy). Three independent experiments were performed.
Anti erk1 2
Manufactured by Merck Group
Sourced in United States, Germany
Anti-ERK1/2 is a lab equipment product that functions as an antibody targeting the extracellular signal-regulated kinases 1 and 2 (ERK1/2). It is used in various research applications to detect and analyze the expression and activation of these important signaling proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho erk1 2, by Merck Group (5 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti akt, by Merck Group (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Anti p erk1 2, by Cell Signaling Technology (2 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Anti phospho p38, by Cell Signaling Technology (2 mentions)
Anti perk1 2, by Merck Group (2 mentions)
Chemoluminescence, by Cytiva (1 mentions)
Anti β tubulin, by Abcam (1 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti p53, by Agilent Technologies (1 mentions)
Anti cleaved caspase 7 asp198, by Cell Signaling Technology (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
24 protocols using anti erk1 2
1
Western Blot Protein Analysis Procedure
2
Western Blot Analysis of Signaling Proteins
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After 24 hours of culture 2 × 106 cells were lysed in MLB buffer (125 mM Tris-HCl, 750 mM NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mM MgCl2, 5 mM EDTA, 25 mM NaF, 1 mM NaVO4, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, pH 7.5), sonicated (with two bursts of 10 s; Labsonic sonicator, Sartorius Stedim Biotech S.A., Aubagne Cedex, France), and centrifuged at 13000 × g for 10 min at 4°C. Ten μg cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membrane sheets (Immobilon-P, Millipore, Billerica, MA, USA) and probed with the following antibodies: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1–2 (Millipore); anti-ERK1–2 (Millipore); anti-phospho-(Ser473)-Akt (Millipore); anti-Akt (Millipore); anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as control of equal protein loading; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), followed by the secondary peroxidase-conjugated antibodies (Bio-Rad). Proteins were detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA).
3
Immunoblot Analysis of CRC Signaling
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Cells of the indicated CRC lines were grown in 24-well plates in complete culture medium until reaching subconfluency before they were treated with SMI for 6 h. Afterwards, protein extracts were prepared and subjected to immunoblot analysis as published before [34 (link)], using polyvinylidene fluoride membrane for protein transfer. The following primary antibodies (all from New England BioLabs, Frankfurt, Germany, unless specified otherwise) were employed: anti-GAPDH (#2118), anti-phospho-AKT (P-AKT; #4060), anti-phospho-MEK (P-MEK1/2; #9154), anti-phospho-ERK1/2 (P-ERK1/2) (#4370), anti-AKT protein (#4691), anti-MEK1/2 (#8727), and anti-ERK1/2 (#06-182, Millipore, Billerica, MA, United States). The blots were developed using LI-COR reagents for an Odyssey Infrared Imaging System as previously described [35 ]. The signal intensities of the investigated proteins were quantified by means of the Odyssey software and raw data processed as described in the corresponding figure legend.
4
3D Culture Protein Extraction and Western Blot
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After 11 days of 3D culture, Matrigel™ was dissolved and the whole-cell extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4, 1 % NP40, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM Na3VO4, 1 mM NaF) with protease and phosphatase inhibitor cocktail (Roche Bâle, Switzerland). Three-dimensional whole-cell extracts (30 μg) or 50 μg of tumoral proteins, extracted by 8 M urea, were resolved by 12 % SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad, Marnes la Coquette, France).
Blots were incubated with anti-phospho-MEK1/2 (1:1 000 dilution, Cell Signaling, Boston, MA, USA), anti-MEK1/2 (1:2 000 dilution, Cell Signaling), anti-phospho-ERK1/2 (1:1 000 dilution, Cell Signaling), anti-ERK1/2 (1:1 000 dilution, Millipore, Darmstadt, Germany), anti-phospho-Akt (1:1 000 dilution, Cell Signaling), anti-Akt (clone 11E7, 1:1 000 dilution, Cell Signaling) or anti-LIN7A (1:1 000 dilution; Thermo Scientific, Rockford, IL, USA) antibodies. Beta-actin (1:10 000 dilution, Sigma Aldrich, Saint-Quentin Fallavier, France) was used as internal control for protein loading. Quantification of the expression was assessed by densitometry analysis.
Blots were incubated with anti-phospho-MEK1/2 (1:1 000 dilution, Cell Signaling, Boston, MA, USA), anti-MEK1/2 (1:2 000 dilution, Cell Signaling), anti-phospho-ERK1/2 (1:1 000 dilution, Cell Signaling), anti-ERK1/2 (1:1 000 dilution, Millipore, Darmstadt, Germany), anti-phospho-Akt (1:1 000 dilution, Cell Signaling), anti-Akt (clone 11E7, 1:1 000 dilution, Cell Signaling) or anti-LIN7A (1:1 000 dilution; Thermo Scientific, Rockford, IL, USA) antibodies. Beta-actin (1:10 000 dilution, Sigma Aldrich, Saint-Quentin Fallavier, France) was used as internal control for protein loading. Quantification of the expression was assessed by densitometry analysis.
5
Automated Capillary Isoelectric Immunoassay
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NanoPro 1000 is an automated capillary-based isoelectric-focusing immunoassay system (ProteinSimple; http://www.proteinsimple.com ). Protein isolation, detection, and quantification were done as per the manufacturer's instructions. Immunoprobing was done using primary antibodies obtained from Cell Signaling Technology (anti-phospho-ERK) and Millipore (anti-ERK1/2), and then probed with horseradish peroxidase–conjugated, goat anti-rabbit secondary antibodies (Jackson ImmunoResearch). All antibody incubation and wash steps were programmed and performed automatically in the NanoPro system; incubation time for primary antibodies was 2 hours and for secondary antibodies 1 hour. A 1:1 digital image was analyzed and quantified with Compass software (ProteinSimple) according to the manufacturer's instructions. Experiments were conducted in triplicates.
6
Investigating TRPM7 and MMP-2 Signaling
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Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), and other cell culture materials were purchased from Gibco Life Technologies Corporation (USA). Anti-TRPM7 antibody (cat# ab85016) was purchased from Abcam (USA). Anti-MMP-2 (cat# 13132), anti-p-Akt (cat# 9271), anti-Akt (cat# 9272), and anti-p-ERK1/2 (cat# 5726) antibodies were obtained from Cell Signaling Technology (USA), while anti-ERK1/2 (cat# 442704) antibody was purchased from Millipore (Canada). Anti-β-actin antibody (cat# A1978) was purchased from Sigma-Aldrich (USA). Pierce BCA Protein Assay Kit was from Pierce Biotechnology (USA). Phenylmethylsulfonyl fluoride (PMSF), sodium chloride (NaCl), and sodium dodecyl sulfate (SDS) were obtained from Bioshop (Canada). All other reagents, unless specified, were from Sigma-Aldrich (USA).
7
Immunoblotting and Quantification of ppErk
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For immunoblotting, cells were lysed in RIPA buffer and lysates were separated on polyacrylamide gels before transfer to nitrocellulose membranes. Antibodies used were anti-ppErk (Sigma, M9692) and anti-Erk1/2 (Millipore, 06-182) at 1:500 dilution. Detection was performed using fluorescently labeled secondary antibodies at 0.1 μg/ml (LI-COR) and scanning in a LI-COR Odyssey system. Intensities of bands were quantified in ImageStudio (LI-COR).
8
Cytokine and Protein Analysis in Astrocyte Cultures
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For cytokine analysis, supernatants from cell culture were removed and examined using ELISA for IL6 (DY406) according to the manufacturer’s instructions (R&D systems) and exactly as we have described before [21 (link)]. For Western blotting, astrocytes were scraped from the culture plate and suspended in PTxE buffer (PBS, 1 % Triton-X, 1 mM EDTA). Samples were denatured and electrophoresis carried out on 10 % SDS-polyacrylamide gels exactly as we have previously reported [21 (link)]. Primary antibodies used were anti-pERK (Millipore, 05-797R), anti-pAKT (Cell Signalling, S473), anti-ERK 1/2 (Millipore 05-481) and anti-actin (Abcam, ab3280). Secondary antibody used was HRP conjugated mouse (Sigma, A8924) or rabbit (GE Healthcare, NA934).
9
Inhibition of SR-B1 Signaling Pathway
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SR-B1 inhibitors BLT-1, ITX-5061 and Glyburide were purchased from Sigma-Aldrich (St Louis, MO), Wuxi Apptech Co (Shanghai, China) and Enzo Life Sciences (Farmingdale, NY), respectively. Mouse interferon α 1, interferon β and interferon gamma were purchased from PBL Biomedical Laboratories (New Brunswick, NJ), Biorbyt (Cambridge, UK) and Immunotools (Friesoythe, Germany). CPZ was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). siRNA was purchased from Ambion, Life Technologies (Austin, TX) and were transfected with TransIT-X2 (Mirus Bio LLC; Madison, WI). Human HDLs were isolated from serum by sequential centrifugation from a healthy volunteer. BSA-FITC, human SAA and human ApoA1 were purchased from Sigma Chemical Co. (St. Louis, MO). L37pA (DWLKAFYDKVAEKLKEAFP), L37pA-FITC, amyloid β (Aβ), LL37 and L37mut (DWLKYKDKLKEKLKEALFP) peptides were purchased from GenScript (Piscataway, NJ). M1 peptide was provided by Dr. Lasarte (CIMA, Spain). For Western blot, anti-phospho STAT1, anti-phospho AKT, anti-AKT, anti-phospho p38, anti-p38 and anti-phospho ERK1/2 were purchased from Cell Signaling Technologies (Beverly, MA). Anti-STAT1 was from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-ERK1/2 was purchased from Millipore (Bedford, MA). Anti-TLR2, anti-TLR4 and anti-IFNAR1 antibodies were purchased from BioLegend (San Diego, CA).
10
Immunoblotting for Gal1 and VEGFR2
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Immunoblotting was performed as described (34 (link)). Briefly, equal amounts of rGal1 or protein extracts from HUVECs (30 µg) exposed to plasma samples preincubated for 1 h with human anti-Gal1 mAbs (clone 21 or 42) or isotype control were resuspended in Laemmli buffer under reducing conditions. Proteins were resolved in 4 to 20% SDS–PAGE and blotted onto nitrocellulose membranes (GE Healthcare). After blocking, the membranes were probed with anti-human Gal1 mAb-21 and mAb-42 (1.5 μg/mL): anti-VEGFR2 (1/1,000; Cell Signaling), anti-phospoho-VEGFR2 (1:1,000; Cell Signaling), anti-ERK-1/2 (1:2,000; Sigma), and anti-phospho-ERK-1/2 (1:1,000; Sigma). Blots were then incubated with horseradish peroxidase (HRP)–labeled anti-human, rabbit, or mouse secondary Ab 1:2,000 (Vector) and developed using the Immobilon Western Chemiluminescent HRP Substrate (Millipore). Protein bands were analyzed with Fiji 2.0 analysis software.
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