Western blot analysis was performed as previously described37 (link). For these experiments, we used a mouse monoclonal antibody against nNOS (1:2000, BD Biosciences), a rabbit polyclonal antibody against nNOS phosphorylated In Ser1417 (P-nNOS, 1:2000, Abcam), a rabbit monoclonal antibody against Rho kinase 1 (ROCK1, 1:1000, Abcam), a mouse monoclonal antibody against ROCK2 (1:500, Abcam), a rabbit polyclonal antibody against myosin phosphatase (MYPT, 1:2000, Abcam), a rabbit polyclonal antibody against MYPT phosphorylated In Thr696 (P-MYPT, 1:500, Abcam), and a monoclonal anti-β-actin-peroxidase antibody (1:50000, Sigma-Aldrich, Spain). Appropriate positive controls were used for each blot.
Anti β actin peroxidase antibody
Manufactured by Merck Group
Sourced in United States, Israel
The Anti-β-actin-peroxidase antibody is a reagent used in immunohistochemistry and Western blotting applications. It is conjugated with horseradish peroxidase (HRP) enzyme and specifically recognizes the β-actin protein, a cytoskeletal protein found in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Rock1, by Abcam (1 mentions)
P mypt, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Ab2740, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Merck Group (1 mentions)
Anti hur antibody 3a2, by Santa Cruz Biotechnology (1 mentions)
Ap0893, by ABclonal (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Trans blot semi dry transfer cell, by Bio-Rad (1 mentions)
Ab23722, by Abcam (1 mentions)
Ab51074, by Abcam (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
17 protocols using anti β actin peroxidase antibody
1
Quantifying Protein Expression via Western Blot
2
Quantifying HCV Protein Expression
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Protein concentrations of the extracts were assayed by Bradford reagent (Bio-Rad) and equal amounts of cell extracts were separated by SDS-12% PAGE and transferred onto a nitrocellulose membrane (Sigma). Samples were then analyzed by western blot using the desired antibodies, anti-HCV core antibody (ab2740, Abcam), anti-HuR antibody (3A2, Santa Cruz and 07–1735, Merck Millipore), anti-pan-p-Ser/Thr antibody (AP0893, Abclonal), anti-pan-p-Tyr antibody (AP0905, Abclonal), anti-PKC-δ antibody (A7778, Abclonal), anti-p-PKC-δ-T505 (AP0776) followed by the respective secondary antibodies (horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG; Sigma). Mouse-monoclonal anti-β-actin- peroxidase antibody (A3854, Sigma) was used as a control for equal loading of total cell extracts. Antibody complexes were detected using the ImmobilonTM Western systems (Millipore).
NS3 antibody used for immunostaining and co-immunoprecipitation was a kind gift from Prof. Guangxiang (George) Luo, University of Alabama.
NS3 antibody used for immunostaining and co-immunoprecipitation was a kind gift from Prof. Guangxiang (George) Luo, University of Alabama.
3
Comparative Analysis of AGE and MT1-MMP in Collagen and Ovarian Cancer Cells
4
Quantitative Protein Analysis via Western Blotting
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Western blot was performed as described previously (13 (link)). Briefly, separated proteins in SDS-PAGE were
transferred onto polyvinylidene fluoride membranes and were sequentially
immune-reacted with specific primary antibodies and secondary antibodies.
Antibody-conjunct proteins were quantified using SuperSignal™ West Pico
Chemiluminescent Substrate (Thermo Scientific, USA). The primary antibodies
cleaved caspase-3 (Cell Signaling Technology (CST, USA)), Bcl-2 (CST), Bax
(CST), Lin28a (CST), Sp-1 (CST), HMGA2 (CST), K-ras (CST), Snail (CST), and
Zcchc11 (Abcam, UK) were applied after the membranes were blocked in either 5%
milk or 5% BSA. Anti-β-actin-peroxidase antibody was obtained from Sigma-Aldrich
(USA) and used as an internal reference. The protein content was analyzed using
Image Lab 5.1 (Bio-Rad, USA).
transferred onto polyvinylidene fluoride membranes and were sequentially
immune-reacted with specific primary antibodies and secondary antibodies.
Antibody-conjunct proteins were quantified using SuperSignal™ West Pico
Chemiluminescent Substrate (Thermo Scientific, USA). The primary antibodies
cleaved caspase-3 (Cell Signaling Technology (CST, USA)), Bcl-2 (CST), Bax
(CST), Lin28a (CST), Sp-1 (CST), HMGA2 (CST), K-ras (CST), Snail (CST), and
Zcchc11 (Abcam, UK) were applied after the membranes were blocked in either 5%
milk or 5% BSA. Anti-β-actin-peroxidase antibody was obtained from Sigma-Aldrich
(USA) and used as an internal reference. The protein content was analyzed using
Image Lab 5.1 (Bio-Rad, USA).
5
Western Blot Analysis of Midgut Proteins
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Midguts were homogenized in 2x Laemmli sample buffer (Bio-Rad), boiled for 5 min, and centrifuged at 10,000 g for 10 min. The supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% non-fat dried milk in a Tris-buffered saline buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, pH 7.5) (TBST) for 1 h, the membrane was incubated in the blocking solution overnight at 4°C with anti-His tag monoclonal antibody (Thermo Scientific) or anti-human collagen IV polyclonal antibodies (Abcam, Cambridge, MA, USA). Following overnight incubation with the primary antibodies, membranes were washed three times (6 min/wash) in TBST. Membranes were incubated with anti-rabbit IgG-HRP or anti-mouse IgG-HRP (Cell Signaling Technology, Danvers, MA, USA) at RT for 2 h and then treated for 1 min with SuperSignal West Pico chemiluminescent substrate (Pierce, Waltham, MA, USA). The immunoreactive proteins were visualized by exposing the membrane to an x-ray film. Anti-β-actin-peroxidase antibody (Sigma) was used to detect the loading control in each lane.
6
Immunohistochemical Analysis of Lung Tumor Markers
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Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of human and mouse lung tumor tissues as previously described [16 (link)]. Primary antibodies include rabbit polyclonal anti-human Oct4 (2750, Cell Signaling, Beverly, MA), rabbit monoclonal anti-human M-CSF (ab52864, Abcam, Cambridge, MA), and rabbit polyclonal anti-human/mouse CD206 (ab64693, Abcam). The immunoreactive intensity was analyzed with the HistoQuest analysis software (TissueGnostics, Vienna, Austria). Immunoblot analysis was performed to detect Oct4 and β-actin (as the loading control) using rabbit polyclonal anti-human Oct4 antibody (2750, Cell Signaling) and mouse monoclonal anti-β-actin-peroxidase antibody (A3854, Sigma-Aldrich, St. Louis, MO), respectively. For ELISA, A549 cells (1 × 106) that had been transduced with recombinant lentiviruses derived from pSin-null, pSin-EF2-Oct4, shLuc, shOct4#4880, or shOct4#4883 lentiviral vectors were cultured in 6-well plates, and their conditioned media were collected after 72 h for quantifying the levels of M-CSF, TNF-α, IL-1β, and IL-6 secretion with ELISA kits (R &D, Minneapolis, MN).
7
Quantitative Protein Expression Analysis
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Total protein lysates were extracted with lysis buffer (1% Triton, 50 nmol/L Tris, 15 mmol/L NaCl) supplemented with protease inhibitors (Sigma–Aldrich, St. Louis, MO, USA). The following primary antibodies were used:: ITGA6 (recombinant rabbit monoclonal antibody, clone: EPR18124, catalog number: ab181551, Abcam), E2F1 (mouse polyclonal antibody, clone: C-20, catalog number: sc-193, Santa Cruz), YY1 (mouse monoclonal antibody, clone: 2E11C5, catalog number: 66281-1-Ig, Proteintech, Manchester, United Kingdom), E2F2 (mouse monoclonal antibody, clone: TFE-25, catalog number: sc-9967, Santa Cruz Biothecnology, Dallas, TX, USA), β-actin (mouse monoclonal Anti-β-actin−Peroxidase antibody, clone: AC-15, catalog number: A3854, Sigma–Aldrich, St. Louis, MO, USA) and vinculin (mouse monoclonal antibody, clone: hVIN-1, catalog number: V9131, Sigma–Aldrich, St. Louis, MO, USA). Proteins were visualized by enhanced chemiluminescence detection system (Sigma–Aldrich, St. Louis, MO, USA). Quantification was performed by Quantity One 4.6.6 software (Bio-Rad, Hercules, California, USA).
8
Quantitative Analysis of RHOA Pathway
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The expression levels of the RHOA protein and MLC phosphorylation by the MLC Kinase part of the RHOA pathway were analysed using western blot in five animals Dp1Yey and five control (wt) littermates (see Fig. 4G , Supplementary Material, Fig. S16 , and Supplementary Information ). We used the following primary antibodies: anti-RHOA (2117, Cell Signalling, USA, 1:1000), anti-pMLC (Thr18/Ser19 #3674, Cell signalling, Boston, MA, USA, 1:1000) and mouse monoclonal Anti-β-Actin-Peroxidase antibody (A3854 Sigma, 1:150 000); and HRP conjugated Goat anti-Rabbit IgG secondary antibody (A16096, Invitrogen, France). Protein signals were visualized with Amersham™ Imager 600 and were quantified using ImageJ and statistical analysis using Sigma Plot. The relative amount of RHOA and p-MLC proteins was calculated as the ratio of the signal detected for each protein of interest compared with the β-actin signal detected and normalized by the mean signal of the wt samples.
9
Antibody Characterization for Signaling
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Antibodies against PLCγ1, p-PLCγ1 (Tyr783), AMPK, p-AMPK (T172), LC3B, mTOR, p-mTOR (Ser2448), FAK, p-FAK (Y397), p-ULK1 (S757), Bcl-2, and Beclin1 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Antibodies targeting IP3R, ULK1, and p62 were purchased from Abcam Inc. (Cambridge, MA, USA). Anti-β-actin peroxidase antibody and the HA-probe were purchased from Sigma-Aldrich in China (Shanghai, China) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Other reagents were of the highest grade commercially available.
10
Western Blot Analysis of iNOS Protein
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Forty (40) μg of protein from whole cell lysate were loaded on 7.5% polyacrylamide-SDS gels and blotted on a nitrocellulose membrane. After blocking with 4% BSA for 90 min, membranes were incubated overnight at 4 °C with rabbit anti-iNOS antibody (130 kDa) (1:500, Cayman Chemicals, Ann Arbor, MI, USA). Upon washing, the blots were incubated for 90 min in the corresponding conjugated donkey anti-rabbit antibody (1:10,000, GE Healthcare, Buckinghamshire, UK). The position of the individual protein was detected after exposure to the ChemiDocTM XRS+ (Bio-Rad Laboratories, Hercules, CA, USA) image system. Band-intensity analysis was performed using a computerized image analysis system (ImageJ software, version 1.40C, NIH). Protein quantity was normalized to β-actin protein (40 kDa) level measurements using mouse monoclonal anti-β-Actin−Peroxidase antibody (1:20,000, Sigma Aldrich, Israel).
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