The drugs were dissolved in DMSO at the final concentration of 10 mM. Imatinib was purchased from Cayman Chemical (Michigan, USA) and used in the range of 5–25 µM; Gemcitabine was obtained from Sigma Aldrich Corp. (St Louis, MO, USA) and used in the range of 1–10 µM; Cisplatin, kindly donated by Prof. Justin Stebbing (Imperial College, London), was used in the range 1–25 µM. The following antibodies were used: MSLN mouse monoclonal (Santa Cruz); β-actin mouse monoclonal (Abcam), p53 mouse monoclonal (Santa Cruz); pERK, mouse polyclonal (Abcam); PARP rabbit polyclonal (Cell Signaling); pAKT rabbit polyclonal (Abcam); ERK1-2 rabbit polyclonal (Abcam); Secondary HRP (horseradish peroxidase)-conjugated goat anti-rabbit IgG and goat antimouse IgG antibodies were from GE Healthcare. The expression plasmid pcDNA3.1 encoding for MSLN (aa 360-2230) was kindly donated by Dr. Uehara (Kansai Medical University, Japan); the empty vector pcDNA3.1, employed as control, was donated by Dr. Giamas (Imperial College, London).
β actin mouse monoclonal
Manufactured by Abcam
Sourced in United States, United Kingdom
β-actin mouse monoclonal is a primary antibody that recognizes the β-actin protein, a commonly used loading control and housekeeping gene. This antibody is produced in mouse and can be used for various applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal p53, by Santa Cruz Biotechnology (1 mentions)
Gemcitabine, by Merck Group (1 mentions)
Imatinib, by Cayman Chemical (1 mentions)
Parp rabbit polyclonal, by Cell Signaling Technology (1 mentions)
Bovine anti goat igg hrp, by Santa Cruz Biotechnology (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Bovine anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mouse monoclonal anti myc 9b11, by Cell Signaling Technology (1 mentions)
Coxiv 3e11, by Cell Signaling Technology (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Pimonidazole, by Hypoxyprobe (1 mentions)
Dasatinib, by Cell Signaling Technology (1 mentions)
Sds page, by Beyotime (1 mentions)
6 protocols using β actin mouse monoclonal
1
Molecular Mechanisms Modulating Cancer Cell Responses
2
Glucocorticoid Receptor Regulation Analysis
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Glucocorticoid receptor expression and phosphorylation state was investigated by western blot analysis as described [13 (link)] using polyclonal antibodies directed against GRα, phospho-GR(Ser203), phospho-GR(Ser211), phospho-GR(Ser226) (Santa Cruz Biotechnology). The levels of protein and β-actin (β-actin mouse monoclonal: Abcam, Cambridge, UK) were used to control for loading. GR subcellular localization was determined by GRα-YFP transfection (described above) and immunocytochemistry against GRα as described [9 (link)].
3
Western Blot Analysis of Hepatocyte Proteins
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Microsomal or cytosolic proteins (30 µg in each lane) of hepatocytes were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes (0.45 µm) using a Trans-Blot ® Turbo™ Transfer System (Bio-Rad, USA). The membranes were blocked in 5 % non-fat dry milk/TBS-Tween-20 for 2 h. For the immunodetection of CYP1A1, CY-P1A2, GSTA and NQO1, the membranes were probed overnight with primary antibodies [CYP1A1 -rabbit polyclonal, 1:1000 (Novus Biologicals, USA), CYP1A2 -mouse monoclonal, 1:1000 (Novus Biologicals), GSTA -goat polyclonal, 1:3000 (Abcam, UK), NQO1 -rabbit monoclonal, 1:3000 (Novus Biologicals)] diluted in TBS-Tween 20 supplemented with 1 % BSA, washed four times with TBS-Tween 20 buffer and probed with complementary secondary antibodies for 1 h [bovine anti-goat IgG-HRP, 1:3000, Santa Cruz Biotechnology (USA), bovine anti-mouse IgG-HRP, 1:10 000, Santa Cruz Biotechnology, bovine anti-rabbit IgG-HRP, 1:10 000, Santa Cruz Biotechnology]. The signal was detected using an enhanced Amersham ECL chemiluminescence kit (GE Healthcare Life Sciences, USA) according to the manufacturer's instructions. β-actin (mouse monoclonal, 1:3000, Abcam) served as the loading control. The intensity of bands was evaluated using a C-DiGit™ Blot Scanner (LI-COR Biotechnology, USA). Protein levels were measured in two independent experiments.
4
Hypoxia-Induced HIF-1α Signaling Pathway
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The rabbit polyclonal CHCHD4 and rabbit polyclonal BNIP3 antibodies were purchased from Cambridge Biosciences. The mouse monoclonal HIF-1α antibody was purchased from BD Biosciences. The mouse monoclonal β-actin and rabbit polyclonal ATP5B antibodies were purchased from Abcam. The mouse monoclonal anti-myc (9B11) and rabbit monoclonal COXIV (3E11) antibodies were purchased from Cell Signaling Technology. The mouse monoclonal anti-Pimonidazole antibody was purchased from HypoxyProbe, Inc. The donkey anti-rabbit and anti-mouse horseradish peroxidase-linked secondary antibodies were purchased from VWR. Sodium azide was purchased from Sigma-Aldrich and used at 5 mM for 16 h in order to optimally block HIF-1α protein levels without significantly affecting cell viability. DAPI solution (10 mg/mL) was purchased from Cambridge Biosciences. Pimonidazole (200 mM) was purchased from HypoxyProbe, Inc.
5
EPHA2 Protein Expression and Phosphorylation
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EPHA2 Monoclonal Antibody (1C11A12) Catalog # 37-4400 (WB analysis for total-EPHA2) was purchased from Thermo Scientific, and mouse monoclonal β-Actin (Catalog # ab8226) was bought from Abcam. Phospho-EPHA2 (pEPHA2 Ser897) (D9A1) Catalog # 6347 and pEphA2 Tyr772 Catalog # 8244S were purchased from Cell Signaling technology. Secondary antibodies such as IRDye® 800CW Goat anti-Rabbit IgG Secondary antibody (Catalog # 926-32211) and IRDye® 680RD Goat anti-Mouse IgG Secondary antibody (Catalog # 926-68070) were purchased from Licor. Inhibitors for EPHA2, such as dasatinib, were bought from Cell Signaling, and ALW-II-41-27 was bought from Sigma-Aldrich.
6
Protein Expression Analysis of HPMC Cells
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HPMCs were collected in each group and fully lysed with radioimmunoprecipitation assay for 15 to 20 min. The cells were centrifuged for 15 min at 12,000 rpm at a low temperature and the protein concentration was quantified by the bicinchoninic acid (BCA) method. The samples of 40 ug were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime) and electro-transferred onto polyvinylidene fluoride (PVDF) film for 120 min at a constant current of 200 mA. Then, the samples were sealed for 1 to 2 h at room temperature in 5% non-fat dry milk/Tris-buffered saline Tween (TBST). The primary antibodies (rabbit monoclonal α-SMA, rabbit monoclonal E-cadherin, rabbit monoclonal Vimentin, rabbit monoclonal PTEN, rabbit monoclonal Wnt3a, rabbit monoclonal β-catenin, rabbit monoclonal CD63, rabbit monoclonal TSG101, and mouse monoclonal β-actin antibodies were obtained from Abcam) were diluted with TBST, and the corresponding bands were incubated overnight from primary antibody dilution buffer at 4°C. The secondary antibodies were incubated for 1 to 2 h at room temperature and bands were developed by the electrochemiluminescence (ECL) method. Experiments were repeated at least 3 times.
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