The following chemicals were used: SRT HCl (ApexBio, #A4180), RES (Sigma-Aldrich, #711004), SA3 (Santa Cruz Biotechnology, #SC-222315), EX527 (Selleckchem, #S1541), and INH (Sigma-Aldrich, #I3377). The following antibodies were used: anti-SIRT1 (Millipore, #07-131; Abcam, #E104), anti–NF-κB p65 (Santa Cruz Biotechnology, #SC-372), anti–NF-κB p65 (acetyl K310) (Abcam, #ab19870), anti-LC3B (Cell Signaling Technology, #3868), anti-CD3 (Dako, #A0452), anti-CD68 (Dako, #M0814), anti-CD163 (Serotec, #MCA1853), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10) (Cell Signaling Technology, #2118), anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)–linked antibody (Cell Signaling Technology, #7074), and mouse anti-rabbit IgG (conformation-specific) monoclonal antibody (Cell Signaling Technology, #3678). Stealth SIRT1 small interfering RNAs (siRNAs) (set of three: #HSS118729, #HSS177403, and #HSS177404) and control siRNAs (#1299001) were from Life Technologies and Integrated DNA Technologies, respectively.
Anti rabbit immunoglobulin g igg horseradish peroxidase hrp linked antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody is a secondary antibody conjugated to the enzyme horseradish peroxidase. It is used to detect and visualize primary antibodies that are raised against rabbit antigens in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh 14c10, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Anti tubulin, by Cell Signaling Technology (2 mentions)
Anti acc, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti mcherry, by Abcam (2 mentions)
Anti atg5, by Cell Signaling Technology (2 mentions)
Anti phospho ampk t172, by Cell Signaling Technology (2 mentions)
Anti phospho acc s79, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Histone h3 antibody, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Anti cd68, by Agilent Technologies (1 mentions)
Anti nf κb p65 acetyl k310, by Abcam (1 mentions)
Anti cd163, by Bio-Rad (1 mentions)
Control sirna, by Thermo Fisher Scientific (1 mentions)
Anti sirt1, by Merck Group (1 mentions)
10 protocols using anti rabbit immunoglobulin g igg horseradish peroxidase hrp linked antibody
1
SIRT1-Mediated Regulation of NF-κB and Autophagy
2
Quantification of Renal Transporter Proteins
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The primary mouse renal tubular epithelial cells and kidney tissues were lysed using the CelLytic MT lysis reagent. Equal amounts of proteins (100 μg/lane) were separated by electrophoresis using 10% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). The membranes were incubated for 1 h in a blocking solution containing 5% nonfat milk in Tris-buffered saline and further incubated for 12 h at 4 °C with the antibodies recognizing OAT1 (1:500; MyBioSource), OAT3 (1:1000; Santa Cruz), GLUT9 (1:800; Invitrogen), URAT1 (1:800; MyBioSource), and β-actin (1:1000; Cell Signaling Technology). Thereafter, the membranes were incubated with secondary antibodies (anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody, 1:3000; Cell Signaling Technology, Inc.) for 1 h at room temperature. The immunoreactive protein bands were detected using EzWestLumi plus (ATTO, Tokyo, Japan) and analyzed using Ez-Capture II (ATTO) and CS Analyzer v.3.0 (ATTO).
3
Western Blotting Analysis of Cellular Proteins
4
Herbal Extract Modulates Cell Apoptosis
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The herbs of XSTQ were obtained from the Pharmacy of Traditional Medicine of Peking University People's Hospital (Beijing, China). The human SH-SY5Y cell line was obtained from the cell resource center of Peking Union Medical College. Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kits were obtained from BD Biosciences (San Jose, California, USA). RNeasy Mini kits were purchased from Qiagen (Valencia, CA, USA). The SuperScript™ III First-Strand Synthesis System was purchased from Invitrogen Corporation (Carlsbad, CA). 2.5 × SYBR Green Real Master Mix was purchased from TIANGEN Biotech Co Ltd (Beijing, China). Nitrocellulose filter membranes were purchased from Whatman (Maidstone, UK), and SuperSignal® West Pico chemiluminescent substrate was purchased from Pierce Biotechnology (Rockford, USA). Antibodies to p-Erk1/2, Erk, p-p38 MAPK, p38 MAPK, p-JNK, JNK and anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody were purchased from Cell Signaling Technology Inc (Danvers, MA, USA).
5
Protein Expression Analysis in Cell Lysates
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Protein samples were prepared by lysing cells with radioimmunoprecipitation assay (RIPA) buffer (Abcam) in the presence of sodium orthovanadate, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride (PMSF), all purchased from Sigma. Protein concentration in the supernatants was done by ultracentrifugation using the Microcon® Centrifugal Filter Devices (Merck Millipore). Protein concentration was determined with DC protein assay (Bio-Rad). Equal amounts of protein were separated with 10% Mini-PROTEAN TGX Gel (Bio-Rad) and then transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with HSP27, HSP70, HSP60, PD-L1, STAT3, phospho-STAT3 (Tyr705), beta- actin (Cell Signaling Technology) antibodies at 1:1,000 and NLRC5 rat mAb (Clone3H8, 1:1,000; Merck) overnight. Primary antibodies were detected with anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology) except for the NLRC5 rat monoclonal antibody which was detected with anti-rat IgG, HRP-linked antibody (Cell Signaling Technology) and developed with SignalFire™ Enhanced Chemiluminescence (ECL) Reagent (Cell Signaling Technology).
6
Immunoblot Analysis of RNase L, Histones, and Cytoskeletal Proteins
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Immunoblot analysis was performed as described in [13 (link)]. Mouse anti-RNase L antibody 2E9 (Novus Biologicals catalog no. NB100-351) was used at 1:1500. Histone H3 antibody (Thermo Fisher Scientific; NB500-171) was used at 1:1000. Mouse anti-alpha tubulin (Abcam: ab18251) was used at 1:1000. Rabbit anti-GAPDH (Cell Signaling Technology: 2118L) was used at 1:2000. Anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)–linked antibody (Cell Signaling Technology: 7074S) was used at 1:3000. Anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology: 7076S) was used at 1:10,000 Crude nuclear and cytoplasmic fractionation was performed as described in [58 (link)].
7
Western Blot Antibody Dilutions
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Immunoblot analysis was performed as described in (9 (link)). Rabbit anti-GAPDH (Cell Signaling Technology: 2118L) was used at 1:2000. Anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)–linked antibody (Cell Signaling Technology: 7074S) was used at 1:3000. Anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology: 7076S) was used at 1:10,000. Rabbit anti-PARP (Cell Signaling Technology: 9452S) was used at 1:1500. Histone H3 antibody (Thermo Fisher Scientific; NB500-171) was used at 1:1000.
8
Western Blotting Analysis of Cellular Proteins
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Cells were lysed in ice-cold RIPA buffer and western blotting was performed as described previously (Florey et al., 2011 (link)). The following antibodies were used: anti-E-cadherin (1:500; 3195, Cell Signal), anti-tubulin (1:2,000; 3873, Cell Signal), anti-Atg5 (1:500; 2630, Cell Signal), anti-phospho-ACC-S79 (1:500; 3661, Cell Signal), anti-ACC (1:500; 3662, Cell Signal), anti-phospho-AMPK-T172 (1:500; 2531, Cell Signaling), anti-mCherry (1:500; ab125096, Abcam), anti-β-actin (1:2000; A1978, Sigma-Aldrich), anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (1:5,000; 7074, Cell Signal), and anti-mouse IgG HRP-linked antibody (1:5,000; 7076, Cell Signal).
9
Investigating KCNJ15 and APAF-1 Pathways
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The following chemicals were used: KCl (Sigma-Aldrich, P954), paraformaldehyde (Electron Microscopy Sciences, 15710). The following antibodies were used: anti-KCNJ15 (Sigma-Aldrich, HPA016702), anti-APAF-1 (Cell Signaling Technology, 5088), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10) (Cell Signaling Technology, 2118), anti-β-actin (Cell Signaling Technology, 4967), anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, 7074). ON-TARGET plus SMARTpool Human KCNJ15 (3772) small interfering RNAs (siRNAs) (L006245000005) and control siRNAs (1299001) were from Dharmacon and Integrated DNA Technologies, respectively.
10
Protein Expression Analysis Protocol
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Primers
of total protein was estimated by using BCA protein assay kit (23225; Thermo Scientific). Protein sample, 20 μg, was separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (3010040001; Roche). Next, 3% non-fat milk dissolved in tris-buffered saline with 0.1% tween-20 (TBS-T) was introduced to block the membrane for 1 h at room temperature. Then the membrane was incubated with primary antibodies at 4°c overnight against retinoid-related orphan receptor-γt (RORγt, 1:1000, 562894; BD Biosciences), Forkhead box P3 (Foxp3, 1:1000, 14-5773; Invitrogen Antibodies), phospho(p)-JAK2 (1:1000, SAB4300124; MilliporeSigma, Germany), JAK (1:1000, 3230; Cell Signaling Technology, USA), phospho(p)-STAT3 (1:1000, 9145; Cell Signaling Technology), STAT3 (1:1000, 9139; Cell Signaling Technology), and β-actin (1:1000, 4970; Cell Signaling Technology). After being washed thrice with TBS-T, the membrane was incubated with anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (1:2000, 7074; Cell Signaling Technology) for 2 h at room temperature. Finally, the specific protein band was detected using Immobilon ECL ultra western HRP substrate (WBULS0100; Millipore). β-actin was regarded as the control protein, and the Gel pro 3.0 software was used to quantify protein level.
of total protein was estimated by using BCA protein assay kit (23225; Thermo Scientific). Protein sample, 20 μg, was separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (3010040001; Roche). Next, 3% non-fat milk dissolved in tris-buffered saline with 0.1% tween-20 (TBS-T) was introduced to block the membrane for 1 h at room temperature. Then the membrane was incubated with primary antibodies at 4°c overnight against retinoid-related orphan receptor-γt (RORγt, 1:1000, 562894; BD Biosciences), Forkhead box P3 (Foxp3, 1:1000, 14-5773; Invitrogen Antibodies), phospho(p)-JAK2 (1:1000, SAB4300124; MilliporeSigma, Germany), JAK (1:1000, 3230; Cell Signaling Technology, USA), phospho(p)-STAT3 (1:1000, 9145; Cell Signaling Technology), STAT3 (1:1000, 9139; Cell Signaling Technology), and β-actin (1:1000, 4970; Cell Signaling Technology). After being washed thrice with TBS-T, the membrane was incubated with anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (1:2000, 7074; Cell Signaling Technology) for 2 h at room temperature. Finally, the specific protein band was detected using Immobilon ECL ultra western HRP substrate (WBULS0100; Millipore). β-actin was regarded as the control protein, and the Gel pro 3.0 software was used to quantify protein level.
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