RARα and PML-RARα protein levels were detected by western blot analysis. HL-60 cells were grown in 6-well plates and treated with the following conditions for 48 hours: 1) DMSO as vehicle control, 2) 12μM JY-1-106, 3) 200nM SR11253, 4) 12μM JY-1-106 plus 200nM SR11253. After treatments, cells were collected by spinning down at 500xg for 10 minutes. Cell pellets were resuspended in 200μL RIPA buffer with protein inhibitor (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack, Roche) and the lysates were centrifuged (17000xg 30 minutes at 4°C). Samples were separated by 4–20% SDS-PAGE and proteins were transferred to Immobilon-FL PVDF membrane (Millipore). Membrane was incubated with Odyssey Blocking Buffer (LI-COR) for 1 hour and then with Rabbit anti RARα antibodies (Biolegend) and β-Actin (8H10D10) Mouse mAb (Cell signaling) for 2 hours at room temperature. The membrane was washed 3 times with TBS-T and then incubated with IRDye 800CW Goat anti-Rabbit IgG (H + L) (LI-COR) and IRDye 680LT Goat anti-Mouse IgG (H + L) (LI-COR) for 1 hour. Images were taken and data were quantified by Odyssey® Fc system from LI-COR.
β actin 8h10d10 mouse mab
Manufactured by Cell Signaling Technology
Sourced in United States
The β-Actin (8H10D10) Mouse mAb is a monoclonal antibody that recognizes the β-actin protein, a highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used for the detection and quantification of β-actin in various applications, including Western blotting, immunocytochemistry, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Easypack, by Roche (2 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey fc system, by LI COR (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Irdye 680lt goat anti mouse igg h l, by LI COR (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase labeled antibodies, by Cell Signaling Technology (1 mentions)
Mekk2, by Cell Signaling Technology (1 mentions)
Sc 13125, by Santa Cruz Biotechnology (1 mentions)
Ab70493, by Abcam (1 mentions)
Ab15246, by Abcam (1 mentions)
Ki 67 8d5 mouse mab, by Cell Signaling Technology (1 mentions)
Gapdh d16h11 rabbit mab, by Cell Signaling Technology (1 mentions)
Secondary hrp conjugated antibodies for western blotting, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Clarity max, by Bio-Rad (1 mentions)
Alliance mini hd9, by Uvitec (1 mentions)
11 protocols using β actin 8h10d10 mouse mab
1
Quantifying RARα and PML-RARα Protein Levels
2
Protein Expression Analysis by Western Blot
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Cells were lysed in cell lysis buffer containing protease and phosphatase inhibitors (Cell Signaling Technologies). Protein concentrations were assessed by BCA assays (Thermo Fisher Scientific) according to the manufacturer’s instructions. About 40 µg of protein was separated on 10% SDS-PAGE gels and electroblotted on PVDF membranes. Nonspecific binding was blocked using 5% bovine serum albumin (BSA) (Sigma) in TBS-Tween-20 (TBST) (Sigma). MAP3K2 (MEKK2), HIF-1α, and COX2 protein were detected using MEKK2 (#19607, Cell Signaling Technology), Purified Mouse Anti-Human HIF-1α Clone 54/HIF-1α (RUO) (BD Biosciences), and Cox-2 (clone H-3, #sc-376861, Santa Cruz Biotech) antibodies. β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling Technology) served as loading control. Antibodies were diluted in TBST with 1% BSA. Immunoreactivity was assessed using horseradish peroxidase-labeled antibodies (Cell Signaling Technology).
3
Immunofluorescence and Western Blotting Protocols
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Antibodies used in this study are as follows: UBF (F-9) mouse monoclonal antibody used for immuno-FISH (sc-13125; Santa Cruz Biotechnology), UBF (M01) mouse monoclonal antibody used for immunofluorescence and Western blotting (clone 6B6; Abnova), c-Myc rabbit polyclonal antibody (5605; Cell Signaling Technology), nucleolin rabbit polyclonal antibody (ab70493; Abcam), GAPDH (D16H11) rabbit mAb (5174; Cell Signaling Technology), RAD21 (D213) antibody (4321; Cell Signaling Technology), β-Actin (8H10D10) mouse mAb (3700; Cell Signaling Technology), Ki-67 (8D5) mouse mAb (9449; Cell Signaling Technology), and α-tubulin antibody (ab15246; 1:500; Abcam). Secondary antibodies for immunofluorescence (Alexa Fluor 488 and 594 conjugates) were obtained from Life Technologies and used at 1:500 dilution. Secondary HRP-conjugated antibodies for Western blotting were from Cell Signaling Technology and typically used at 1:5,000 dilution.
4
Western Blot Analysis of Protein Targets
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Total protein was extracted using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA, pH 8.0) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and quantified using the Bradford method. Proteins were separated on gradient SDS-PAGE gels and transferred to nitrocellulose membranes (Whatman). Membranes were then incubated with the primary antibodies overnight at 4 °C. The following primary antibodies were used: MeCP2 (D4F3) XP Rabbit (#3456, Cell Signaling Technology); β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling Technology); PPARγ (81B8) Rabbit (#2443, Cell Signaling Technology). After incubation with the specific HRP-conjugated antibody (Vector; 1:10,000 dilution), the chemiluminescent signal was detected using Clarity and/or Clarity Max (Bio-Rad, Italy) and images were acquired with Alliance Mini HD9 (Uvitec, Cambridge, UK). Densitometric analysis was performed with ImageJ software (https://imagej.nih.gov/ij/download.html ). Full and uncropped Western Blots were provided as Supplemental Material.
5
Biomarkers and Signaling Pathway Analysis
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Blood urea nitrogen (BUN), serum triglyceride, and serum total cholesterol were performed with a biochemical analyzer (Roche, COBAS C311). Serum creatinine (Scr) was examined with Creatinine Assay Kit (sarcosine oxidase) purchased from Nanjing Jian Cheng Bioengineering Institute (C011‐2‐1). Mouse Anti‐dsDNA Ig's (Total A + G + M) ELISA Kits (Catalog # 5110) were provided by Alpha Diagnostic International. Immunoglobulin G (IgG) (Total) Mouse Uncoated ELISA Kits (Catalog # 88‐50400‐88) were purchased from Invitrogen. Anti‐Rubicon/Baron antibody (ab156052), anti‐NOX2/gp91phox antibody (ab80508), and Goat‐anti‐Mouse IgG H&L (Alexa Fluor® 488) (ab150113) were from Abcam. LC3A/B (D3U4C) XP® Rabbit mAb (#12741S), and β‐Actin (8H10D10) Mouse mAb (#3700) were from Cell Signaling Technology. 10 × RIPA Lysis Buffer (20‐188, Merck Millipore) was used for Western Blot. Rubicon, Nox2, and LC3 proteins were normalized to β‐actin. Urine was collected by properly pressing on the bladder of mice at a single time point and urine protein was measured semiquantitatively using urine dipsticks (Global Biotech Co., Ltd).16
6
NOS1 Deficiency in Mice and LPS-induced Inflammation
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6 to 8 weeks old WT and NOS1−/− C57BL/6 J mice were obtained from the Jackson Laboratory. The study was approved by the ethics board of Chongqing University of Posts and Telecommunications (CQUPT) (No. CA2019-01). Animal handling and experiments were conducted in accordance with the policies of the Animal Care Facility of CQUPT. LPS from Escherichia coli O111:B4 was obtained from Sigma. IL4 was purchased from PeproTech. The RNeasy Mini Kit was purchased from Qiagen, and the High Capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix were obtained from Applied Biosystems. The STAT6 (C9) mouse monoclonal antibody (mAb) was purchased from Santa Cruz Biotechnology. β-Actin (8H10D10) mouse mAb, NF-κB p65 (D14E12) XP® rabbit mAb, iNOS (D6B6S) rabbit mAb, PPARγ (C26H12) rabbit mAb, and Phospho-Stat6 (Tyr641) (D8S9Y) rabbit mAb were obtained from Cell Signaling Technology. The SOCS1 rabbit antibody (ab62584) was purchased from Abcam.
7
Western Blot Analysis of SOD1 and SOD2
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Western blotting (WB) analysis was performed on 40 μg lysates from young and old myoblasts and myotubes, using SOD1 (71G8) mouse mAb (number 4266, Cell Signalling Technology, Danvers, MA, USA) at 1 : 1000, SOD2 (D9V9C) rabbit mAb (number 13194, Cell Signalling Technology) at 1 : 1000, and β-actin (8H10D10) mouse mAb (number 3700, Cell Signalling Technology) at 1 : 1000, as primary antibody, and secondary HRP-conjugated antibodies (Cell Signalling Technology) at 1 : 5000. Bands were detected and pictured at Bio-Rad GelDoc by LiteAblot PLUS enhanced chemiluminiscent substrate (EuroClone); densitometry analyses were performed with ImageJ software.
8
Western Blot Analysis of Lipid Metabolism Proteins
9
Apoptosis Signaling Pathway Analysis
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SBC-3 cells (cell concentration: 5
× 105 cells/mL) were cultured in a six-well flat-bottom
plate and treated with EtOH/H2O (1:1), 10 μM cisplatin,
or 15 μM1 for 24 h. The Western blotting analysis
was carried out by the same procedures as previously reported.26 (link) The following antibodies were recruited: β-actin
(8H10D10 Mouse mAb, product number 3700, 1:1000; Cell Signaling Technology,
Danvers, MA, USA), caspase-8 (1C12 Mouse mAb, product number 9746,
1:1000; Cell Signaling Technology), caspase-9 (C9 Mouse mAb, product
number 9508, 1:2000; Cell Signaling Technology), caspase-3 (3G2 Mouse
mAb, product number 9668, 1:1000, Cell Signaling Technology), PARP
(46D11 Rabbit mAb, product number 9532, 1:1000; Cell Signaling Technology),
Bax (2D2 Mouse mAb, product number 89477, 1:1000; Cell Signaling Technology),
Bcl-2 (124 Mouse mAb, product number 15071, 1:1000; Cell Signaling
Technology), anti-rabbit IgG, horseradish peroxidase (HRP)-linked
antibody (product number 7074, 1:10000; Cell Signaling Technology),
and anti-mouse IgG, HRP-linked antibody (product number 7076, 1:10000;
Cell Signaling Technology). The signals were detected using ECL Prime
Western Blotting Detection Reagents (GE Healthcare, Boston, MA, USA),
and photographed by a LAS-3000 luminescent image analyzer (FUJIFILM,
Tokyo, Japan).
× 105 cells/mL) were cultured in a six-well flat-bottom
plate and treated with EtOH/H2O (1:1), 10 μM cisplatin,
or 15 μM
was carried out by the same procedures as previously reported.26 (link) The following antibodies were recruited: β-actin
(8H10D10 Mouse mAb, product number 3700, 1:1000; Cell Signaling Technology,
Danvers, MA, USA), caspase-8 (1C12 Mouse mAb, product number 9746,
1:1000; Cell Signaling Technology), caspase-9 (C9 Mouse mAb, product
number 9508, 1:2000; Cell Signaling Technology), caspase-3 (3G2 Mouse
mAb, product number 9668, 1:1000, Cell Signaling Technology), PARP
(46D11 Rabbit mAb, product number 9532, 1:1000; Cell Signaling Technology),
Bax (2D2 Mouse mAb, product number 89477, 1:1000; Cell Signaling Technology),
Bcl-2 (124 Mouse mAb, product number 15071, 1:1000; Cell Signaling
Technology), anti-rabbit IgG, horseradish peroxidase (HRP)-linked
antibody (product number 7074, 1:10000; Cell Signaling Technology),
and anti-mouse IgG, HRP-linked antibody (product number 7076, 1:10000;
Cell Signaling Technology). The signals were detected using ECL Prime
Western Blotting Detection Reagents (GE Healthcare, Boston, MA, USA),
and photographed by a LAS-3000 luminescent image analyzer (FUJIFILM,
Tokyo, Japan).
10
Breast Cancer Cell Signaling Pathway
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Human MDA-MB-231 cells (mammary gland epithelial cells from breast adenocarcinoma; ATCC HTB-26) were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (R10). AHCC ® was generously provided by Amino Up Co. Ltd. (Sapporo, Japan). The primary antibodies used were EphA2 (D4A2) XP ® rabbit mAb, JNK1 (2C6) mouse mAb, JNK2 (56G8) rabbit mAb, phospho-SAPK/JNK (Thr183/Tyr185) rabbit antibody, NFAT1 (D43B1) XP ® rabbit mAb, JunD (D17G2) rabbit mAb, β-Actin (8H10D10) mouse mAb and β-Actin (13E5) rabbit mAb (all from Cell Signaling Technology, Danvers, MA, United States). The secondary antibodies were horseradish peroxidase (HRP)-conjugated affinity purified goat anti-mouse IgG (H + L) antibody (Proteintech Group Inc., Rosemont, IL, United States) and HRP-conjugated affinity purified goat antirabbit IgG (H + L) antibody (CSL). All antibodies were used at 1:1000-2000 dilutions for primary antibodies and at 1: 2000-5,000 dilutions for secondary antibodies.
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