For Western blotting, BMDMs were seeded in 12-well plate and incubated overnight. These cells were treated with TCMD in the presence or absence of RANKL. At the indicated time point after treatment, cells were lysed in a buffer containing 1% NP-40, 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 Mm Na3VO4, 0.02% NaN3, 2 mM dithiothreitol plus protease inhibitor (Roche, Mannheim, Germany) and phosphatase inhibitor (Sigma-Aldrich). Whole cell lysates were separated using SDS-PAGE and transferred onto nitrocellulose membranes The membranes were probed with primary antibodies against IκBα, phospho-form of p38, ERK, JNK, and p65 (Cell Signaling Technology, Danvers, MA, USA). A primary antibody for β-actin was obtained from Santa Cruz. The membranes were then washed with TBST and incubated with HRP-conjugated secondary antibodies (Santa Cruz, CA, USA) for 2 h at room temperature. The bands were visualized using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) on a luminescent image analyzer (Amersham Imager 600, GE Healthcare, UK).
Primary antibody for β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Canada
This primary antibody for β-actin is a widely used tool for the detection and quantification of the β-actin protein, a cytoskeletal protein found in all eukaryotic cells. The antibody specifically binds to β-actin, allowing researchers to measure its expression levels in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Amersham imager, by Cytiva (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thomas Scientific (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Il 6 elisa kit, by Dakewe (1 mentions)
γh2ax ser139, by Cell Signaling Technology (1 mentions)
Tnf α, by Dakewe (1 mentions)
Goat anti rabbit igg h l, by EarthOx (1 mentions)
A23187, by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
7 protocols using primary antibody for β actin
1
Immunoblotting Analysis of TCMD-Treated BMDMs
2
Western Blot Analysis of Stem Cell Markers
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Monolayer cultures of respective cell lines at 80–90% confluence were lysed using 100 μl of RIPA buffer (Thomas Scientific Inc. Swedesboro, NJ). Tris-glycine (Bio-Rad, Irvine, CA) gels were loaded with 100 μg of total proteins. After electrophoresis, the gel was transferred to a nitrocellulose membrane for 1 hour.
The membrane was blocked for 30 min in 5% skim milk at room temperature. The membrane was briefly rinsed with 1xTTBS and incubated overnight with the respective primary antibodies at 4°C. Primary antibodies of STAT3, pSTAT3, NF-κB, pNF-κB, CD44, and Oct-4 were purchased from Cell Signaling Technology (Danvers, MA). Primary antibody for β-actin was purchased from the Santa Cruz Biotech (Santa Cruz, CA). After incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP), the protein bands were developed with the chemiluminescent reagents.
The membrane was blocked for 30 min in 5% skim milk at room temperature. The membrane was briefly rinsed with 1xTTBS and incubated overnight with the respective primary antibodies at 4°C. Primary antibodies of STAT3, pSTAT3, NF-κB, pNF-κB, CD44, and Oct-4 were purchased from Cell Signaling Technology (Danvers, MA). Primary antibody for β-actin was purchased from the Santa Cruz Biotech (Santa Cruz, CA). After incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP), the protein bands were developed with the chemiluminescent reagents.
3
Paeonol Attenuates UV-Induced Damage
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The SUV lambs used in this study were purchased from Q-Lab Corporation (Cleveland, OH). The percentage of UVA and UVB emitted from SUV lamps was measured by a UV radiometer and was recorded as 92.5% and 7.5%, respectively. Paeonol (the purity >99%) was purchased from Chengdu Ruifensi Biotechnology Co. Ltd (Chengdu, China). The pGEX-GST-H2AX plasmid was purchased from Addgene Inc. The active TOPK was purchased from Millippore Company (Billerica, MA, USA). Both the TNF-α and IL-6 ELISA kits were purchased from Dakewe Biotech Co. Ltd (Shenzhen, China). The primary antibodies for TOPK, JNKs, p38, H2AX, p-TOPK (Thr9), p-JNKs (Thr183/Tyr185), p-p38 (Thr180/Tyr182), and γ-H2AX (Ser139) were purchased from Cell Signaling Technology (USA). The primary antibody for β-actin was obtained from Santa Cruz (USA). Horseradish peroxidase (HRP)-conjugated Goat anti Mouse IgG (H+L) and Goat anti Rabbit IgG (H+L) secondary antibodies were purchased from Earth Ox life sciences company (San Francisco US).
4
Exploring Cell Signaling Pathways
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Dulbecco’s modified Eagle medium (DMEM) was purchased from Hyclone (MD, USA), primary antibodies anti-phosphory-p38 (1:1000), anti-cleaved caspase3 (1:1000), anti-cleaved PARP (1:1000), anti-Bax (1:1000), anti-p38 (1:1000), anti-caspase 3 (1:1000) were purchased from Cell Signaling Technology (MA, USA). Primary antibodies for PCNA and crystal violet dye were purchased from Bioss Biotechnology (Beijing, China). Primary antibody for β-actin and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The flow cytometry reagent was purchased from BD Bioscience (San Jose, CA, USA). The p38 MAPK inhibitor SB203580, DIM (catalog no. BML-GR207), BAPTA-AM, A23187, Fluo-3/AM and Hoechst 33342 dye, were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 and DIM were dissolved in dimethyl sulfoxide (DMSO) as stock solution, respectively. The final concentration of DMSO did not exceed 0.1%.
5
Western Blot Analysis of Cell Cycle Regulators
6
In Vitro and In Vivo ADR Studies
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ADR was supplied by Actavis Italy S.p.A. (Viale Pasteur, Nerviano, Italy). A stock solution of ADR (2 mM) was prepared with DMSO and stored at -20°C for in vitro testing. It was further diluted with DMSO before use. A stock solution of ADR (1 mg/mL) was dissolved with normal saline (NS) and stored at 4°C for in vivo experiments. It was freshly diluted with normal saline before use. Insulin glargine injection (insulin) was obtained from Sanofi-Aventis Deutschland Gmbh. AICAR and streptozotocin were supplied by Sigma-Aldrich and compound C was from Merck & Co. RPMI 1640 medium (no glucose) was supplied by Gibco-BRL (Gaithersburg, MD). The primary antibodies for γH2A.X (Ser139), p-AMPKα (Thr172), AMPK, acetyl-CoA carboxylase (ACC) and p-ACC (Ser79), cleaved PARP and cleaved caspase-3were obtained from Cell Signaling Technology. The primary antibody for β-actin and secondary antibodies for mouse IgG, goat IgG and rabbit IgG were purchased from Santa Cruz Biotechnology. Enhanced chemiluminescence (ECL), a Western blot detection reagent, was obtained from Pierce Chemical.
7
Quantitative Analysis of TGFBI Protein
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The primary antibody for β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TGFBI antibody was purchased from Abcam (Cambridge, UK). Western blotting was performed by loading the whole cell lysate (30 µg) of each sample on a 10% polyacrylamide gel for electrophoresis. The membrane was blocked in 5% skimmed milk in 1x Tris buffered saline solution (pH 7.4) containing 0.05% Tween-20 and used at a concentration of 1:1,000 (for TGFBI) or 1:2,000 (for β-actin). The concentration of the secondary antibody was 1:10,000 to 1:20,000. Pierce Chemical (Rockford, IL, USA)’s Super Signal West Pico or SuperSignal Femto Chemiluminescent Substrate was used to visualize proteins.
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