Western blotting and immunostaining were performed as described previously57 (link),58 (link). Briefly, cells were washed in PBS and lysed in a radioimmunoprecipitation assay buffer containing complete Protease Inhibitor Cocktail and PhosSTOP (Roche). The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).
β actin rabbit antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The β-actin rabbit antibody is a specific antibody that recognizes the β-actin protein, a widely expressed and highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used as a loading control for Western blotting experiments to normalize protein expression levels across samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti sp1, by Cell Signaling Technology (1 mentions)
Phosstop, by Roche (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Blocking one p solution, by Nacalai Tesque (1 mentions)
Sybr green pcr master mix, by Promega (1 mentions)
Rat igg2a isotype control pe, by Thermo Fisher Scientific (1 mentions)
Rabbit ho 1 antibody, by Abcam (1 mentions)
Tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Amv transcriptase, by Promega (1 mentions)
Znppix, by Merck Group (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Rnasin, by Promega (1 mentions)
Alt and ast detection kits, by Nanjing Jiancheng (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Paclitaxel taxol, by Merck Group (1 mentions)
13 protocols using β actin rabbit antibody
1
Western Blotting and Immunostaining Protocol
2
Hepatoprotective Effects of MF
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MF (C19H18O11, FW = 422.34, purity ≥ 95%) was purchased from Nanjing ZeLang Medical Technology Co. Ltd. (Nanjing, China). LPS (Escherichia coli, 0111: B4), D-GalN, and ZnPP IX were obtained from Sigma (St. Louis, MO, USA). ALT and AST detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL, USA). Trizol reagent was purchased from Invitrogen (Grand Island, NY, USA). AMV transcriptase, RNasin, SYBR green PCR Master Mix, and the Dual-Luciferase Reporter Assay kit were obtained from Promega (Madison, WI, USA). The tumor necrosis factor (TNF)-α ELISA kit was obtained from Bender MedSystems (Vienna, Austria). Rabbit HO-1 antibody was obtained from Abcam (Cambridge, MA, UK), rabbit β-actin antibody was purchased from Cell Signaling Technology (Boston, MA, USA), and the rat anti-mouse TLR4/MD-2 complex and rat IgG2a isotype control PE were obtained from eBioscience (San Diego, CA, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA).
3
Colon Cancer Cell Lines and Protocols
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Human normal colon cell lines, CRL-1790 and CCD-18co, and CRC cell lines, HT29, HCT116, DLD-1, and LoVo, were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). The cells were cultured in RPMI medium (Hyclone, MA, USA) with 10% fetal bovine serum (FBS, Gibco, MA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, MA, USA). Cells were maintained in a humidified incubator at 37 °C in an atmosphere with 5% carbon dioxide. The HCT-116 Taxol-resistant (TR) colon cancer cell line was established by exposing cells to increased concentrations of Taxol for 3 months to select surviving cells. Rabbit Fbxw7 antibody was purchased from Sigma-Aldrich (#AB10620, Shanghai, China). Rabbit Nox1 antibody was purchased from Abcam (#ab78016, Shanghai, China). Rabbit β-actin antibody was purchased from Cell Signaling Technology (#4970, Danvers, MA, USA). Paclitaxel (Taxol) and 2-DG were purchased from Sigma-Aldrich (Shanghai, China).
4
Quantitative Analysis of mRNA and Protein
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Total RNA was extracted using TRIzol reagent (Invitrogen). For mRNA analysis, cDNA was synthesized using the TaqMan Reverse Transcription Reagents kit (TaKaRa). Real-time quantitative PCR analysis was performed with different primer sequences using SYBR Green Master Mix (TaKaRa). The primers used for RT-PCR were shown in Table 1. The cells proteins were isolated, quanti ed, and separated via SDS-PAGE. Western blotting was performed as described previously [22] . Primary antibodies included the rabbit β-Actin antibody (Cell Signaling Technology), and the SLC17A9 antibody (Abcam). Anti-rabbit secondary antibodies (Cell Signaling Technology) were used at a dilution of 1:6000.
5
Apoptotic Signaling in Mouse Brain
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To investigate the presence of apoptotic signals, tissue of the right temporal lobe of sampled mice brains was homogenized in RIPA lysis buffer (Beyotime) and Pierce Protease and Phosphatase Inhibitor Mini Tablets (Thermo Scientific), and centrifuged (13,000 g for 15 minutes at 4°C). Protein concentrations were determined using BCA Protein Assay Kits (Thermo Fisher Scientific). An equal amount of protein samples (40 μg/lane) were loaded onto a 12% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore). The membranes were blocked with 5% milk powder and incubated overnight at 4°C with the primary antibodies: Bcl-2 Rabbit Antibody (1:1000, Cell Signaling Technology #3498) and Bax Rabbit Antibody (1:1000, Cell Signaling Technology #2772) and β-actin Rabbit Antibody (1:10000, Cell Signaling Technology #4970). The PVDF membranes were then incubated with the HRP-linked secondary antibody (1:5000, Santa Cruz Biotechnology) at RT for 1 h. Protein bands were visualized using chemiluminescence reagent kit (Amersham Bioscience). The relative densities of the bands were quantified using ImageJ software (NIH).
6
Murine Macrophage Activation Assay
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The following were used in the study: collagenase and Dulbecco Modified Eagle medium (DMEM) cell culture medium (Gibco, USA), Lipofectamine™ 2000 (Invitrogen, USA), IL28RA rabbit antibody (Sigma, USA), FITC rat anti-mouse CD206 (MMR) antibody (Biolegend, USA), APC rat anti-mouse CD206 (MMR) antibody (Biolegend, USA), FITC rat anti-mouse CD11b antibody (Biolegend, USA), rat anti-mouse CD107b (Mac-3) antibody (Biolegend, USA), β-Actin rabbit antibody (Cell Signaling, USA), phospho-AKT (pAKT) (Ser 473) rabbit antibody (Cell Signaling, USA), Arginase 1 (Arg 1) goat antibody (Santa Cruz, USA), PI3KCG mouse antibody (Santa Cruz, USA), Donkey F (ab)2 Anti-Rat IgG H&L(Alexa Fluor® 568) preadsorbed (Abcam, UK), peroxidase-conjugated AffiniPure rabbit anti-goat IgG(H+L) (Jackson, USA), goat anti-rabbit IgG-HRP (Absin Bioscience Inc., China), and peroxidase-conjugated AffiniPure goat anti-mouse IgG(H+L) (ZSGB-BIO, China).
The healthy C57BL/6 mice (6-8 weeks old, 16-20 g weight) were from the Jiangsu Province Animal Center. The lncRNA260-specific siRNA was synthesized by Shanghai Gene Pharma Co., Ltd. The nucleotide sequence is listed inTable 1 .
The healthy C57BL/6 mice (6-8 weeks old, 16-20 g weight) were from the Jiangsu Province Animal Center. The lncRNA260-specific siRNA was synthesized by Shanghai Gene Pharma Co., Ltd. The nucleotide sequence is listed in
7
Western Blot Analysis of FOXL2 Protein
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WB was performed with total cell lysate using antibody specific for FOXL2 (Abcam, ab5096), β-actin rabbit antibody (Cell Signaling Technology, 064967S). Membranes were incubated with a 1:2000 dilution of HRP-conjugated antibody rabbit to goat IgG (Abcam; ab97100) and anti-rabbit IgG (Cell Signaling Technology; 7074P2) and developed with the ECL system.
8
Anti-inflammatory Effects of Lomerizine
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Lomerizine was purchased from Absin Bioscience Inc. (Shanghai, China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, United States). Dexamethasone was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Hematoxylin and eosin (H&E) staining kit was purchased from Abcam (Waltham, Boston, United States). Dulbecco’s Modified Eagle Medium (DMEM) and macrophage colony-stimulating factor (M-CSF) was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Penicillin-streptomycin solution (Pen Strep) was purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). Fetal Bovine Serum (FBS) was purchased from Cegrogen Biotech (Wupperweg, Germany).
Primers were synthesized by Hua Gene Biotech Co., Ltd. (Shanghai, China). The primary antibody against IκB-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibodies against iNOS, phospho-NF-κB p65 (Ser536) Rabbit mAb, NF-kappaB p65 Rabbit mAb, phospho-p38 MAPK (Thr180/Tyr182) XP Rabbit mAb, p38 MAPK XP Rabbit mAb, phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb, SAPK/JNK Antibody, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Antibody, p44/42 MAPK (ERK1/2) Rabbit mAb, and β-Actin Rabbit Antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).
Primers were synthesized by Hua Gene Biotech Co., Ltd. (Shanghai, China). The primary antibody against IκB-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibodies against iNOS, phospho-NF-κB p65 (Ser536) Rabbit mAb, NF-kappaB p65 Rabbit mAb, phospho-p38 MAPK (Thr180/Tyr182) XP Rabbit mAb, p38 MAPK XP Rabbit mAb, phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb, SAPK/JNK Antibody, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Antibody, p44/42 MAPK (ERK1/2) Rabbit mAb, and β-Actin Rabbit Antibody were purchased from Cell Signaling Technology (Danvers, MA, United States).
9
Amyloid-Beta Immunodetection Assay
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We purchased the following antibodies and reagents from the respective manufacturers listed below. A mouse monoclonal antibody to Aβ (6E10) (SIG-39300, Covance, CA, USA); a rabbit polyclonal antibody to APP, C-terminal (A8717, Sigma-Aldrich, MO, USA); a β-actin rabbit antibody (#4970, Cell signaling, MA, USA); a goat anti-mouse IgG-horseradish peroxidase (HRP) antibody (sc-2005, Santa Cruz Biotechnology, CA, USA); a goat anti-rabbit IgG-HRP antibody (sc-2054, Santa Cruz Biotechnology); Alexa Fluor 488 Goat anti-rabbit IgG (H + L) (A11088, Life Technologies, CA, USA); Human β Amyloid (1–40) ELISA kit (292-62301, Wako, Osaka, Japan); Human β Amyloid (1–42) ELISA kit (298-62401, Wako); Protein G-Agarose (11719416001, Roche, Basel, Switzerland); hygromycin B (400052, Calbiochem, CA, USA); SuperSignal West Femto Maximum Sensitivity Substrate (#34095, Thermo Fisher Scientific, Rockford, USA); SuperSignal ELISA Femto Maximum Sensitivity Substrate (#37075, Thermo Fisher Scientific); Ham’s F-12 medium (17458-65, Nacalai Tesque, Kyoto, Japan); Penicillin-Streptomycin (15140-122, Gibco, CA, USA); and fetal bovine serum (SH30910.03, Hyclone, MA, USA).
10
Western Blot Analysis of PHB1 Expression
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