Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose culture medium was purchased from Hyclone (GE Healthcare Life Science, NJ, USA), and fetal bovine serum (FBS) was purchased from Biological Industry (Göttingen, Germany). Dimethyl sulfoxide (DMSO), 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), and bovine serum albumin (BSA) were purchased from Solarbio (Shanghai, China). SP600125 and 5-FU were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies specific for c-Jun (#380397), phospho-c-Jun (Ser63) (#382955), phospho-JNK (Thr183/Tyr185) (#619878), and b-actin (#200068-6D7) were obtained from Zen-Bio (Chengdu, China). Antibodies specific for ABCG2 (#27286-1-AP), PDCD4 (Cat# 12587-1-AP), horseradish peroxidase (HRP)-conjugated AffiniPure Goat anti-rabbit immunoglobin G (IgG) (H+L) (#SA00001-2), HRP-conjugated AffiniPure Goat anti-mouse IgG (H+L) (#SA00001-1), and GAPDH (#60004-1-Ig) were purchased from Proteintech Group, Inc (Rosemont, IL, USA). JNK antibody (#CY5490) was purchased from Abways Technology (China). SP600125 and 5-FU were dissolved in DMSO.
β actin
Manufactured by ZenBio
Sourced in China, United States
β-actin is a housekeeping protein found in all eukaryotic cells. It is a component of the cytoskeleton and plays a crucial role in various cellular processes, including cell motility, structure, and division.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by ZenBio (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Phospho sqstm1 thr269 ser272 specific antibody, by PhosphoSolutions (2 mentions)
Flag tag (flag), by ProSpec (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti myosin, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Sp600125, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
Sds page kit, by Beyotime (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti lc3, by Proteintech (1 mentions)
Il 1β, by ZenBio (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ferritin, by MyBioSource (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
30 protocols using β actin
1
Evaluating JNK Pathway in Anticancer Drug Response
2
HepG2 Protein Extraction and Western Blot Analysis
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HepG2 protein was extracted using the SDS-PAGE kit (Beyotime Biotechnology, Shanghai, China). Protein concentrations were determined using a BCA protein assay kit (Shanghai Fuheng Biotechnology). The protein was then separated using SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking it for one hour with 5% skim milk powder, the primary antibody (anti-LC3, Proteintech Group, and b-actin, Zen-Bio) was added and shaken overnight at 4 °C. Secondary antibody (Zen-Bio) was then added and cells were subjected to agitation at room temperature for one hour. The chemiluminescence assay was carried out using the ECL kit (Shanghai Biyuntian Biotechnology) and detected by ChemiDoc XRS+ (Bio-Rad). Data were analyzed with the ImageJ program.
3
Protein Expression Analysis in Mouse Paw Tissues
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Protein samples of mice paw tissues were lysed with RIPA buffer and protein was separated in the 12% SDS-polyacrylamide gel electrophoresis (PAGE). The blots were then transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were incubated with interleukin-1β (IL-1β) (1:1000, #511369, Zen BioScience, China) or β-actin (1:5000, #250132, Zen BioScience, China) dilutions in TBS containing 1% skim milk. The blots were then incubated with secondary antibodies, HRP-conjugated goat anti-mouse IgGs (Zhongshanjinqiao Co. China) at 1:2000. The experiments were repeated three times with different samples. The blot was cut prior to hybridization with antibodies during blotting. Each blotting gel with 12 loading wells was cut into two equal pieces. Thus, the full-length of the blotting gel included 6 sample-loading wells.
4
Brain Western Blot Analysis
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Western blot (WB)analysis was performed as previously described [20 ]. Briefly, brains were removed immediately after anesthetization and decapitation were performed. Samples were collected from the object region of brains and lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma). Then protein extract was obtained after centrifugation. Equal amounts of protein (40 mg) were loaded onto each lane of SDS-PAGE gels. After gel electrophoresis, proteins were transferred onto nitrocellulose membranes (Millipore) and blocked in nonfat milk at room temperature for 2 h. Then membranes were incubated in primary antibodies at 4 °C for 24 h and incubated in appropriate secondary antibodies at room temperature for 1 h subsequently. The following antibodies were used: Iba-1(Wako), iron regulatory protein 1 (IRP1, ZenBioScience), divalent metal transporter 1 (DMT1, MyBioSource), transferrin receptor1 (TfR1, ZenBioScience), ferritin (MyBioSource), ferroportin1 (Fpn1, MyBioSource), β-actin (ZenBioScience) and β-tubulin (ZenBioScience). Protein bands were visualized using nickel-intensified DAB solution, and band densitometry analysises were performed using Image J software (National Institutes of Health). The housekeeping proteins β-actin or β-tubulin were used as a loading control.
5
Protein Extraction and Western Blot Analysis
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Protein extraction was conducted using RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Thermo Fisher Scientific, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay was performed as in previous reports.52 (link) Primary antibodies used in this study were β-actin (cat. 200068-8F10; ZEN-bioscience), H3 (cat. ASO71; ABclonal), Cyclin D1 (cat. 55506 T; CST), CDK4 (cat. 12790 T; CST), Cyclin A2 (cat. ab181591; Abcam), Cyclin E1 (cat. 20808 S; CST), CDK1 (cat. ab32094; Abcam), Cyclin B1(cat. 55004-1-AP; Proteintech), H3K9me1 (cat. A2358; ABclonal), H3K9me2 (cat. A2359; ABclonal), H3K9me3 (cat. ab176916; Abcam), MDIG (cat. 12214-1-AP; Proteintech), OTX2 (cat. 13497-1-AP; Proteintech), Myc (cat. 10828-1-AP; Proteintech), BRCA2 (cat. AF7817; Affinity), Junb (cat. 10486-1AP; Proteintech), E2F2 (cat. AF4100), ATAD5 (cat. Bs-7656R; Bioss). Western blots were quantified using ImageJ software (Version 1.51n, National Institutes of Health, Bethesda, MD, https://imagej.net/ij/index.html ), as previously described.53 (link) The original and uncropped films of Western blots were provided as Supplementary Figs. S19 –S21 .
6
Western Blot Protein Analysis
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The total protein extract was prepared by homogenizing the cells in 1 × SDS sample buffer. All the protein samples were boiled for 5 min and then resolved in SDS-PAGE. The protein samples separated by SDS-PAGE were transferred to the Immunobilon-FL PVDF membrane (Merck Millipore, IPFL00010). After blocking with 5% non-fat milk in TBST (TBS, pH 7.4, 0.1% Tween-20), the membrane was incubated with primary antibody at 4 ℃ overnight. The membrane was washed and incubated with secondary antibodies in the dark for 2 h. The image was acquired by Li-Cor Odyssey Clx Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). The following primary and secondary antibodies were used for western blotting assay: SQSTM1 (Proteintech, 18420-1-AP), ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); Phospho-SQSTM1 (S403)-specific antibody (Cell Signaling Technology, 39786); FLAG tag (Prospec, ANT-146); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). Dylight 680, or Dylight 800-conjugated secondary antibodies (Thermo Fisher Scientific, A28183, A27042, 35518). See Additional file 1 : Table S2 for further details and dilutions of all antibodies.
7
Protein Fractionation and Immunoblotting
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The total protein extract was prepared by homogenizing the cells in a 1× SDS sample buffer. The NP-40-soluble and -insoluble protein fractions from the cells were prepared as previously described [34 (link)]. Immunoprecipitation and western blot were carried out as previously described [34 (link)]. Primary antibodies against the following proteins were used for western blot analysis: K48-linked Ub chain-specific antibody (Millipore, 05-1307); K63-linked Ub chain-specific antibody (Abcam, ab179434); ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); SQSTM1 (Santa Cruz Biotechnology (sc-28359); ATG5 (Cell Signaling Technology, 12994); LC3B (Cell Signaling Technology, 3868); ATG16L1 (Abcam, ab187671); WIPI2 (Abcam, ab105459); Beclin1 (Proteintech, 11306-1-AP); GFP (Rockland, 600-101-215); FLAG tag (Prospec, ANT-146-b); Myc Tag (Biolegend, MMS-150R); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). See Supplementary Table S2 for further details and dilutions of all antibodies.
8
Western Blot Analysis of Cell Signaling
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Total proteins from samples were extracted using RIPA buffer containing a protease inhibitor cocktail (Roche, 5 892 791 001). The protein concentrations were determined using a BCA kit (Beyotime, P0010s). After electrophoresis, proteins were transferred to PVDF membranes (Millipore, E1078). PVDF membranes were blocked and incubated with corresponding primary antibodies, including p16 (Santa Cruz, sc1161), p21 (Santa Cruz, sc817), p53 (Santa Cruz, sc126), nuclear lamina protein LMNB1 (lamin B1; Beyotime, AF-1408) and β-actin (Zen Bio, 700 068), at 4°C overnight, followed by incubation with secondary antibodies for 1 h at room temperature. Enhanced chemiluminescence detection was performed after the membrane was washed.
9
Western Blot Analysis of Antioxidant Proteins
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Extraction of cell proteins was accomplished by applying RIPA lysis buffer (Beyotime, China), and the total protein concentration was measured by the Enhanced BCA Protein Assay Kit (Beyotime, China). The proteins were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After being blocked in 5% skim milk solution, the membranes were incubated with primary antibodies overnight at 4 °C. The bands were incubated with antibodies specific for CAT (1:1 000, Abcam, USA), Nrf2 (1:1 000, CST, USA), KEAP1 (1:1 000, CST, USA), NQO1(1:1 000, Abcam, USA) and HO-1 (1:4 000, Abcam, USA) overnight at 4 °C. Histone H3 (1:3 000, Abcam, USA), β-Actin (1:1 000, ZenBio, China) and GAPDH (1:1 000, ZenBio, China) were used as the internal control. The PVDF membranes were incubated with secondary antibodies at room temperature. Subsequently, the chemiluminescent reagent (Merck Millipore, USA) and imaging system (LAS4000M, USA) were used to visualize the target protein bands. Finally, the bands were quantified by ImageJ software (NIH, USA).
10
Oxidized mtDNA Activates TBK1-IRF3 Signaling
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Mitochondrial DNA isolated from EG7 cells was suspended in sterile H2O and treated with 75 Gy X-ray irradiation to generate oxidized mtDNA.24 (link) BMDCs derived from WT mice were stimulated with 5 μg/ml mtDNA after direct irradiation or 25 μg/ml DMXAA (5,6-dimethylxanthenone-4-acetic acid; Sigma; positive control)38 (link) for 3 h. Proteins were extracted with radioimmunoprecipitation assay buffer (RIPA; Beyotime) containing proteinase inhibitors (Millipore) and phosphatase inhibitors (KeyGen). Briefly, 30 μg of protein was electrophoresed in 12.5% SDS-PAGE gels and transferred to Immobilon-FL membranes (Millipore). The blots were incubated with antibodies specific for pTBK1 (Ser172), total TBK1, pIRF3 (Ser396), total IRF3 (Cell Signaling Technology) and β-actin (Zen BioScience). Quantitative analysis was performed with ImageJ software.
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