AEC II were incubated with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) for 15 min on ice, then centrifuged at 13,000 × g for 5 min at 4°C. A total of 20 µg protein from cell lysate was separated by 12% SDS-PAGE. Wet gel system was used to transfer protein samples to a PVDF membrane. Following 5% slim milk blocking at room temperature for 1.5 h, the membrane was incubated with a primary anti-caspase 3 antibody (cat. no. sc-7148; 1:500 dilution; Santa Cruz Biotechnology, Inc.,) at 4°C overnight and subsequently incubated with a secondary antibody conjugated to horseradish peroxidase (cat. no. sc-2004; 1:2,000 dilution; Santa Cruz Biotechnology, Inc.). β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.,) was used as an internal control. Protein images were observed with an electrochemiluminescence solution (Pierce; Thermo Fisher Scientific, Inc.). The experiment was repeated three times.
Secondary antibody conjugated to horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Secondary antibody conjugated to horseradish peroxidase is a laboratory reagent used in various immunoassay techniques. It functions as a detection agent, binding to the primary antibody and facilitating the visualization of target analytes through the enzymatic activity of horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Enhanced chemiluminescence kit, by Cytiva (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Electrochemiluminescence solution, by Thermo Fisher Scientific (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Gapdh g 9, by Santa Cruz Biotechnology (1 mentions)
Cyclin d1 a 12, by Santa Cruz Biotechnology (1 mentions)
Image station 4000r, by Kodak (1 mentions)
Gapdh or β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitors and phosphatase inhibitor, by Roche (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti actin monoclonal antibody, by Merck Group (1 mentions)
Model 375a, by GE Healthcare (1 mentions)
α tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection system, by Keygen Biotech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
18 protocols using secondary antibody conjugated to horseradish peroxidase
1
Caspase-3 Expression in AEC II Cells
2
Western Blot and Immunofluorescence Analysis of Tumor Proteins
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Cell lysates were prepared by lysing 2 × 106 cells in lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% NaN3) containing protease inhibitor cocktail tablets (Roche Diagnostics,). Equal amounts of protein were separated on 10% SDS–polyacrylamide mini-gels and transferred to Immobilon PVDF membranes (Millipore, Boston, MA, USA). After blocking in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin, the membranes were incubated with the appropriate primary antibody, followed by a secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). The signals were visualized with western blotting substrates.
For immunofluorescence detection, tumor tissue sections were fixed in 4% paraformaldehyde at room temperature for 5 min, washed with PBS twice, incubated with 1% BSA at 37 ◦C for 30 min to block nonspecific interactions, and then stained with primary antibodies to CD68 (8ug/ml), TLR4(5ug/ml), Bcl6 (2ug/ml), IPMO, and HMGB1(5ug/ml) for 1 h (Santa Cruz Biotechnology or R&D system). Sections were then incubated in rhodamine-labeled goat anti-mouse secondary antibody (Proteintech Group, Inc., Chicago, USA) at room temperature for 1 h. Nuclei were then stained with 4, 6-diamidino-2-phenylindole.
For immunofluorescence detection, tumor tissue sections were fixed in 4% paraformaldehyde at room temperature for 5 min, washed with PBS twice, incubated with 1% BSA at 37 ◦C for 30 min to block nonspecific interactions, and then stained with primary antibodies to CD68 (8ug/ml), TLR4(5ug/ml), Bcl6 (2ug/ml), IPMO, and HMGB1(5ug/ml) for 1 h (Santa Cruz Biotechnology or R&D system). Sections were then incubated in rhodamine-labeled goat anti-mouse secondary antibody (Proteintech Group, Inc., Chicago, USA) at room temperature for 1 h. Nuclei were then stained with 4, 6-diamidino-2-phenylindole.
3
Western Blot Analysis of Cellular Proteins
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Proteins were extracted from subconfluent cultures of cells and then characterised by western blot analysis. Cells were lysed in lysis buffer, resolved on a sodium dodecyl sulphate-polyacrylamide electrophoresis gel, and transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk in phosphate buffered saline containing 0.05% Tween (PBST) for 1 h at room temperature, and then probed with a primary antibody overnight at 4 °C. After extensive washing, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (1:10,000; Santa Cruz Biotechnology Inc.) for 1 h at room temperature. Blots were developed using ECL (PE LifeScience, Waltham, MA, USA).
The following antibodies were used: CDK4 (DCS-35) (1:2000, sc-23896; Santa Cruz), CDK6 (B10) (1:1000, sc-7961; Santa Cruz), Cyclin B1 (G11) (1:200, sc-166757; Santa Cruz), Cyclin D1 (A12) (1:200, sc-8396; Santa Cruz), HDAC2 (C-8) (1:200, sc-9959; Santa Cruz), GAPDH (G-9) (1:3000, sc-365062; Santa Cruz), XIAP (1:1000, 10037-2-lg; proteintech), RICTOR (1:1000, 27248-1-AP; proteintech), p300 (3G230/NM-11) (1:1000, ab14984; Abcam), and YY1 (1:5000, 66281-1-lg; proteintech). Uncropped western blot scans from main blots are displayed in Figs.2c , 3c , 5e , 6 , and Supplementary Figs. 4b and 7 .
The following antibodies were used: CDK4 (DCS-35) (1:2000, sc-23896; Santa Cruz), CDK6 (B10) (1:1000, sc-7961; Santa Cruz), Cyclin B1 (G11) (1:200, sc-166757; Santa Cruz), Cyclin D1 (A12) (1:200, sc-8396; Santa Cruz), HDAC2 (C-8) (1:200, sc-9959; Santa Cruz), GAPDH (G-9) (1:3000, sc-365062; Santa Cruz), XIAP (1:1000, 10037-2-lg; proteintech), RICTOR (1:1000, 27248-1-AP; proteintech), p300 (3G230/NM-11) (1:1000, ab14984; Abcam), and YY1 (1:5000, 66281-1-lg; proteintech). Uncropped western blot scans from main blots are displayed in Figs.
4
Western Blot Analysis of SIRT1 and SIRT3
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Total protein was isolated from arterioles or ASMCs by homogenization in cold RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (Roche). Equal amounts of proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking in 5% bovine serum albumin (BSA) in Tris-buffered saline/Tween-20 (50 mM Tris, pH 7.5, 500 mM sodium chloride, and 0.05% Tween-20) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against SIRT1 or SIRT3 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with a secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) for 1 h at room temperature, immunoreactivity signals were detected by chemiluminescence using ECL detection reagent (Advansta Westernbright ECL, USA), visualized using a chemiluminescence detection system (Image Station 4000R, KODAK, USA), and analyzed using Quantity One software (version 4.52).
5
Rab35 Expression in Neurodegenerative Models
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The substantia nigra (SN) and striatum (ST) tissues were collected from wild-type mice, MPTP-treated mice, rotenone-treated mice, SCA3 transgenic mice, (R1441C) LRRK2 or (G2019S) LRRK2 transgenic mice. Briefly, the SN or ST tissues were homogenized with CHAPS lysis buffer containing protease inhibitors (protease inhibitor cocktail, Sigma-Aldrich). A total of 30 μg proteins was separated by 12% SDS-polyacrylamide gel and transferred to PVDF membrane. The membranes were blocked in 5% low-fat milk solution in phosphate-buffered saline with 0.05% Triton X-100. Then, the membranes were hybridized with anti-Rab35 antibody followed by incubation with secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX). Immunoreactive proteins were visualized by using an enhanced chemiluminescence kit (GE Biosciences). To confirm equal amount of protein sample loading, membranes were stripped and reblotted with monoclonal anti-actin antibody (Millipore). Gel bands were quantified by a densitometer (Molecular Dynamics Model 375A) and normalized to an actin control band. Anti-Rab35 polyclonal antibody and monoclonal anti-α-synuclein antiserum were purchased from ProteinTech, Inc (Chicago, IL). The antibody against actin (clone C4) was obtained from Millipore (Darmstadt, Germany).
6
Intestinal Mucosa Protein Analysis
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Intestinal mucosa samples were homogenized with lysis buffer for 30 seconds in a mortar and pestle with liquid nitrogen. Homogenates were centrifuged at 13000 rpm for 10 min at 4°C and the supernatant was collected as the source of protein sample. The proteins were processed with standard methods for Western blot analysis as described [10 (link)]. Rat monoclonal anti-tryptase antibody, rat monoclonal anti-gp91phox and anti-p47phox antibodies, rat monoclonal anit-P-selectin and anti-ICAM-1 antibodies, and α-tubulin antibody were obtained from Santa Cruz (Santa Cruz, CA, USA). The secondary antibody conjugated to horseradish peroxidase was diluted at 1 : 2,000 (Santa Cruz, USA). Immunoblots were incubated with an enhanced chemiluminescence detection system (KeyGen Biotech, China) and the densitometry analysis was performed using Quantity One software.
7
Protein Expression Analysis via Western Blot
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Cells were lysed using cell lysis buffer as previously reported [13 (link)]. Nuclear and cytoplasmic extracts were prepared as previously reported [14 (link)]. Equivalent amounts of protein were separated by SDS-PAGE (8–12 %) and transferred onto polyvinylidene fluoride membranes (Millipore, Danvers, MA, USA). The membranes were blocked and subsequently incubated with the following primary antibodies: anti-p57 antibody (1:500), anti-phospho-cofilin (Ser3) antibody (1:300, 11139, Signalway Antibody, Pearland, TX, USA), anti-β-actin antibody (1:1000, sc-130301, Santa Cruz) and anti-lamin A antibody (1:600, 613501, Biolegend, San Diego, CA, USA). Blots were visualized with a secondary antibody conjugated to horseradish peroxidase (Santa Cruz) and an ECL detection system (Millipore). Western blotting was repeated three times for each protein.
8
Senescence-Associated p21 and p53 Expression
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Total protein was extracted from young (P3) and senescent (P16) RPESCs (1 x 106 cells/sample) as described previously [16 (link)], with some modifications. Membranes were incubated overnight with the primary p21 and p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using GAPDH as the endogenous control, followed by incubation with the secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Protein detection on the membrane was performed using the Clarity Western ECL Substrate Kit (Bio-Rad). The signals were captured with an Alliance Mini (UVITEC Cambridge, Cambridge, UK) system; the p21 and p53 bands were quantified with UVITEC software and their intensity was normalized by comparison to the housekeeping protein β-actin, used as a loading control. The intensity of each band was compared to the negative controls and any change was expressed as a percentage.
9
Western Blot Analysis of TGR5 and PLC-ε
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Lysates were prepared from STC-1 cells and were separated by SDS-PAGE followed by transfer onto nitrocellulose membranes (Immobilon-FL, Millipore, Billerica, MA). The membranes were blocked, incubated with antibodies to TGR5 (1:1000) or PLC-ε (1:1500) (Abcam, Cambridge, MA; Proteintech, Chicago, IL), and following washing, incubated with secondary antibody conjugated to horseradish peroxidase (Santa Cruz biotechnology, Santa Cruz, CA). Proteins on the membrane were detected by the enhanced chemiluminescence detection system (Amersham Life Science Buckinghamshire, UK).
10
Western Blot Analysis of Caspase-1
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