Protein lysates and western blotting were performed as described earlier (Hill et al., 2019 (link)). β-actin mouse monoclonal antibody (Cat. No. A5441, Sigma, St. Louis, MO) was used at 1:3000 dilution. Anti-rabbit secondary antibody from Millipore (Cat. No. AP307P, Darmstadt, Germany, 1:3000 dilution) and anti-mouse secondary antibody (Cat. No. 1706516, Bio-Rad, Hercules, CA) were used at 1:3000 dilution (Fardi et al., 2019 (link)). The protein bands were detected by chemiluminescence and quantified using Bio-Rad Image Lab 5.2.1 analysis software (Arun et al., 2018 (link)).
Anti rabbit secondary antibody
Manufactured by Merck Group
Sourced in United States, Germany
The anti-rabbit secondary antibody is a laboratory reagent used in various immunoassay techniques. Its core function is to specifically bind and detect the presence of primary antibodies raised against rabbit antigens. This allows the visualization and quantification of target analytes in samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Anti mouse secondary antibody, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ecl chemiluminescent detection reagent, by GE Healthcare (3 mentions)
Chemiluminescence substrate, by Merck Group (2 mentions)
Fusion solo station, by Vilber (2 mentions)
β actin antibody, by Cell Signaling Technology (2 mentions)
Cy3 labeled rabbit secondary antibody, by Merck Group (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Bio dot apparatus, by Bio-Rad (2 mentions)
Nucleospin tissue, by Macherey-Nagel (2 mentions)
Luminol, by Santa Cruz Biotechnology (2 mentions)
Ecl advance, by GE Healthcare (2 mentions)
Anti polyglutamate chain, by Adipogen (2 mentions)
Z devd fmk, by Santa Cruz Biotechnology (2 mentions)
Anti mouse, by Merck Group (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Fusion sl 3500, by Vilber (2 mentions)
63 protocols using anti rabbit secondary antibody
1
Western Blot Analysis of Protein Lysates
2
Western Blot Analysis of Protein Expression
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Protein expression was analyzed with a specific antibody. Equal amounts of protein extract (40 μg, nuclear or total extract) were boiled for 6 min in sample buffer [300 mM Tris-HCl (pH 6.8), 3.85% DTT, 9% sodium dodecylsulfate (SDS), 25% glycerol, and 0.033% bromophenol blue], subjected to 8%∼12% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were probed with first antibodies against γ-GCSh (1: 500), actin (1: 104), COX-2 (1: 500), Bcl-2 (1: 250), GRP78 (1: 1000), ATF4 (1: 250), ATF6α (1: 250), Survivin (1: 1000), pro-caspase 3 (1: 1000), PARP-1 (1: 1000), β-catenin (1: 1000) and SP-1 (1: 500). After washing in TBST buffer, the membrane was further incubated with anti-rabbit secondary antibody (Chemicon, Millipore, Billerica, MA, USA) for 1-h. Detection was performed by enhanced chemiluminescence (ECL; Millipore, Billerica, MA, USA) after incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. β-actin was used as the loading control.
3
Western Blot Analysis of Protein Samples
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The cells were harvested into the sample buffer (125 mM Tris–HCl pH 6.8, 20% glycerol, 4% SDS, 5% β-mercaptoethanol, and 0.02% bromophenol blue). The samples were resolved by SDS–PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane, incubated with the primary antibodies (see the list of antibodies in Table S2) and with an anti-rabbit secondary antibody (Merck), and visualized by chemiluminescence substrate (Merck) using a Fusion Solo station (Vilber).
Table S2
4
Protein Expression Analysis by Western Blot
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Cells were seeded overnight, and we transfected plasmids into cells via Lipofectamine 2000 reagent (Life Technologies, USA). 48 h post-transfected, cells were incubated in mammalian cell lysis reagent (Thermo Scientific, USA) with protease inhibitor cocktail (Roche, Switzerland) for 20 mins at 4 °C, centrifuged at 13000 g for 15 mins, the supernatant was harvested as the total protein. Next, 70 µg total protein per lane was loaded on 10% SDS-PAGE gel. And protein bands were transferred from gel to PVDF membranes at 70 V pressure for 1 h, blocked overnight with 5% non-fat milk at 4 °C, followed by sequential incubation with primary antibodies and secondary antibodies. Antibodies used were as follows: TCRP1 antibody (Santa Cruz Biotechnology, 1:300), c-Myc antibody (Cell Signaling Technology, 1:4000), β-actin antibody (Cell Signaling Technology, 1:4000), and anti-rabbit secondary antibody (Merck, 1:10000). Finally, we used chemiluminescent detection system (Millipore, USA) to detect signal.
5
Western Blot Protein Detection
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The cells were harvested into the sample buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 5% βmercaptoethanol, 0.02% bromophenol blue). The samples were resolved by SDS-PAGE, transferred onto a PVDF membrane, incubated with the primary antibodies (see the list of antibodies in Table S2) and with an anti-rabbit secondary antibody (Merck), and visualized by chemiluminescence substrate (Merck) using a Fusion Solo station (Vilber).
6
Western Blot Analysis of TRIB2 Signaling
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The cells were harvested and lysed in RIPA lysis buffer (Sigma Aldrich) supplemented with a protease and phosphatase inhibitor cocktail (Sigma Aldrich). Protein extracts were run on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by voltage gradient transfer. The blots were blocked for 1 h at room temperature in 5% non-fat dry milk. The membranes were then incubated overnight at 4 °C with the primary antibodies for TRIB2 (ATLAS ANTIBODIES, Bromma, Sweden), for AKT, Phospho-Akt (Ser473), ERK, and Phospho-ERK (137F5) (Cell Signaling Technology, Danvers, MA, USA) diluted at 1:1000, and then incubated with an anti-rabbit secondary antibody (Sigma-Aldrich). The visualization of TRIB2 protein bands was obtained by using the horseradish peroxidase (HRP) development system.
7
Histological and Cellular Analysis of Immune Cell Phenotypes
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For the histological portion of the study, paraffin-embedded sections were stained with hematoxylin and eosin. Toluidine blue or Giemsa staining was used to detect mast cells or neutrophils, respectively. The stained cells were counted in 5 different areas, and average positive numbers were calculated on mm2 per skin area.
For cell polarization and AQP9 expression, cells were cultured on glass coverslips and stimulated with fMLP (Sigma), CCL19 (R&D, MN, USA), or CCL21 (R&D, MN, USA). Cells were fixed with 4% formalin in PBS, permeabilized with 0.1% saponin, and stained with phalloidine-Alexa488 (Invitrogen), and AQP9 (Alpha diagnostic international, San Antonio, Texas, USA), anti-rabbit secondary antibody (FITC, Sigma). Frozen LN sections were stained with anti-Ly6G/Alexa594 (clone 1A8, BioLegend) and anti-CD8/FITC (clone 16-10A1, eBioscience).
For cell polarization and AQP9 expression, cells were cultured on glass coverslips and stimulated with fMLP (Sigma), CCL19 (R&D, MN, USA), or CCL21 (R&D, MN, USA). Cells were fixed with 4% formalin in PBS, permeabilized with 0.1% saponin, and stained with phalloidine-Alexa488 (Invitrogen), and AQP9 (Alpha diagnostic international, San Antonio, Texas, USA), anti-rabbit secondary antibody (FITC, Sigma). Frozen LN sections were stained with anti-Ly6G/Alexa594 (clone 1A8, BioLegend) and anti-CD8/FITC (clone 16-10A1, eBioscience).
8
Quantification of miR-206 and Cx43 Expression
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Messenger RNA expression of microRNA-206 and connexin 43 by quantitative real-time polymerase chain reaction Total RNA was extracted from tissues or cells with TRIzol reagent (Invitrogen, USA). For qPCR, RNA was reversely transcribed to cDNA from 1 mg of total RNA using a Reverse Transcription Kit (Takara, Japan). qRT-PCR was conducted with SYBR® premix Ex Taq™ II (perfect real-time) (Takara). All protocols were performed according to the manufacturer's instructions. Results were normalized to the expression of U6/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and then expressed as a relative expression compared with related control samples. The primer sequences were listed in Table 1 .
Protein expression of connexin 43 by Western blotting assay Total proteins were extracted with cell lysis solution, and the concentration was determined by Bradford method. About 50 μg protein was separated in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to PCDF membrane, and incubated with rabbit anti-human Cx43 antibody (1:1000, Santa, USA) at 37°C for 4 h, with anti-rabbit secondary antibody (1:2500, Sigma, USA) at 37°C for 2 h. Protein bands were quantified from film by densitometry using Adobe Photoshop V7.01 imaging system.
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9
Autophagy Modulation in Cancer Therapeutics
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Metformin (D150959), CQ (C6628), MDC (30432), 3-MA (M9281), 4',6-diamidino-2- phenylindole (DAPI; D9542), antibodies against anti-β and LC3, anti-rabbit secondary antibody, anti-mouse secondary antibody and Cy3-labeled rabbit secondary antibody were purchased from Sigma (St. Louis, MO, USA). Antibodies against p-Stat3 (Y705), Stat3, cyclin D1, LC3, PCNA and PARP were purchased from Cell Signaling (Beverly, MA, USA); antibodies against p-AMPK, p-mTOR, Beclin-1, Bcl-2, p62 and Bax were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-phospho-Histone H3 (Ser10) antibody was purchased from Upstate (Millipore, Billerica, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Gibco (Life Technologies Gibco/BRL, New York, NY, USA). Crystal violet staining and JC-1 were obtained from Beyotime Institute of Biotechnology (Shanghai, China). ApopTag plus peroxidase in situ apoptosis detection kit was obtained from Millipore. AO was obtained from Poly-Sciences (Warrington, PA, USA). Stat3, AMPK, Beclin-1 and Atg5 siRNAs were purchased from Shanghai GenePharma (Shanghai, China). Lipofectamine 2000 was bought from Invitrogen (Carlsbad, CA, USA).
10
Detecting Redox Proteins via Western Blot
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For Western blot analysis, 5 μg of total protein from soluble extracts were separated by 12% acrylamide SDS-PAGE gels, transferred to nitrocellulose membranes (Bio-Rad), and probed with AnNTRC (1:1000), 2-Cys Prx (1:5000) and TrxA (thioredoxin A) (1:3000) antibodies. In order to detect High Molecular Mass (HMM) species 10% acrylamide non-reducing SDS-PAGE (where reducing agents were avoided in the loading buffer) and native-PAGE were used. Signals were detected using an anti-rabbit secondary antibody (Sigma-Aldrich, St Louis, MO, US) and the ECL-Plus immunoblotting detection system (GE Healthcare, Buckinghamshire, England).
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