A total of 50 μg of proteins from either cell lysate or articular cartilage homogenate were loaded onto a 12% SDS-PAGE gel. After transfer, membranes were blocked by 5% non-fat milk and incubated with different primary antibodies for overnight at 4°C. Next day, corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated. After washing, the membranes were finally carried out with SuperSignal1 West Pico Chemiluminescent Substrate Kit (Thermo Scientific, Waltham, MA, USA). Rabbit anti-MMP1 (1:2,000), rabbit anti-MMP3 (1:2,000), rabbit anti-MMP9 (1:2,000), rabbit anti-MMP13 (1:2,000), rabbit anti-ADAMTS4 (1:2,000), rabbit anti-ADAMTS5 (1:2,000), mouse anti-GAPDH (1:1,000), rabbit anti-IκBa (1:2,000), rabbit anti-p- IκBa (1:2,000), rabbit anti-p65 (1:2,000), rabbit anti-p-p65 (1:2,000), goat anti-mouse immunoglobulin G H&L (HRP) (1:3,000), and goat anti-rabbit immunoglobulin G (HRP) (1:3,000) were purchased from Abcam (Cambridge, MA, USA). The western blot results were quantitated and analyzed using GS-900 Calibrated Densitometer and software Image Lab (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions.
Rabbit anti p65
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-p65 is a primary antibody that specifically recognizes the p65 subunit of the NF-κB transcription factor. It can be used in various immunological techniques to detect and study the p65 protein.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (1 mentions)
Rabbit anti p p65, by Abcam (1 mentions)
Gs 900 calibrated densitometer, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Goat anti rabbit igg conjugated to horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Rabbit anti iκbα, by Cell Signaling Technology (1 mentions)
Rabbit anti nik, by Cell Signaling Technology (1 mentions)
Rabbit anti acetylated histone h3, by Merck Group (1 mentions)
Mouse anti gapdh, by HyTest (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p p65, by Cell Signaling Technology (1 mentions)
Rabbit anti cd36, by Abcam (1 mentions)
7 protocols using rabbit anti p65
1
Western Blot Analysis of Cartilage Degradation
2
Immunoblot Analysis of NF-κB Pathway
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Western blot analysis was performed as described previously using the following antibodies: mouse anti-phosphorylated IκBα (Cell Signaling, Danvers, MA, USA), rabbit anti-IκBα (Cell Signaling), rabbit anti-acetylated histone H3 (Millipore, Billerica, MA, USA), rabbit anti-NIK (Cell Signaling), mouse anti-p100/p52 (Millipore), rabbit anti-phosphorylated p65 (Cell Signaling) and rabbit anti-p65 (Abcam, Cambridge, MA, USA). Mouse anti-α-Tubulin (Sigma) and mouse anti-GAPDH (HyTest, Turku, Finland) were used as loading controls. Goat anti-mouse IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) as secondary antibodies and enhanced chemiluminescence were used for detection (Amersham Bioscience, Freiburg, Germany). Alternatively, infrared dye-labeled secondary antibodies and infrared imaging were used for detection (Odyssey imaging system, LI-COR Bioscience, Bad Homburg, Germany). Representative blots of at least two independent experiments are shown.
3
Western Blot Analysis of AMPK Signaling
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Proteins were extracted from the hepatocytes and liver tissues using RIPA lysis buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein concentration was quantified using the BCA reagent (Thermo Fisher Scientific) following the manufacturer's protocol. An equal amount of protein from each sample was loaded into each lane for separation by SDS-PAGE and then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking with 5% (w/v) skim milk powder dissolved in PBS containing Tween-20 (PBST) at room temperature for 2 hours, the membranes were incubated at 4 °C overnight with the primary antibodies: rabbit anti-Prkab1 (Proteintech Group), rabbit anti-Prkaa1 and Prkaa2 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pAMPK (Cell Signaling Technology), rabbit anti-ACC pan (Abcam), rabbit anti-pACC (Abcam), rabbit anti-CD36 (Abcam), rabbit anti-p65 (Abcam), rabbit anti-p-p65 (Cell Signaling Technology) and mouse anti-β-actin (Abcam). After washing with PBST, the membranes were incubated at room temperature for 1 hour with the horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Abcam). The density of each band was quantified by densitometric analysis with Image Lab 6.0 software.
4
NF-kB Activation by AvrA Proteins
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HeLa cells were transiently transfected with 500 ng pCMV-HA-avrA(SE), pCMV-HA-avrA(SP), or pCMV-HA plasmids. After 24 h cultivation followed by stimulation with 15 ng/mL TNF-α for 30 min, the cells were fixed for 20 min in 4% paraformaldehyde in PBS, washed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and washed again. Fixed samples were incubated in blocking solution (5% BSA in PBS) for 2 h at 37 °C followed by overnight incubation with rabbit anti-p65 (Abcam, Cambridge, UK) and mouse anti-HA (Abcam, Cambridge, UK). The samples were then incubated with goat anti-rabbit IgG (Alexa Fluor® 488; Abcam, Cambridge, UK) and goat anti-mouse IgG (Alexa Fluor® 546, Cambridge, UK) for 5 h and DAPI for 20 min. The coverslips were mounted on glass slides, and stained cells were observed by laser scanning confocal microscopy (Leica Microsystems, Mannheim, Germany).
5
Evaluating NF-κB p65 Expression in RAW264.7 Cells
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Immunofluorescence staining of RAW264.7 cells was carried out to evaluate the level of protein expression and the distribution of NF-κB p65. We seeded 1 × 10 3 cells per well in 24-well plates containing glass coverslips. The test cells were subjected to treatment with GLPS for 24 h. After the treatment and removal of medium, we fixed the cells with 4% paraformaldehyde for 15 min and then permeabilized them in 0.1% Triton X-100 (Solarbio, China) for 10 min. Subsequently, nonspecific binding sites were blocked at RT for an hour in 5% bovine serum albumin (BSA) buffer (Solarbio, China). After rinsing three times with PBST (PBS containing 0.1% Tween-20), the cells were incubated with mouse anti-CD86 (diluted 1 : 200, Santa Cruz, USA), goat anti-CD206 (diluted 1 : 200, RD, USA), and rabbit anti-P65 (diluted 1 : 200, Abcam, UK) overnight at 4 degrees Celsius. Then, we stained the cells with the corresponding secondary FITC-conjugated antibodies (diluted 1 : 200, Proteintech, USA) or PE-conjugated antibodies (diluted 1 : 200, Proteintech, USA) at RT for an hour in the dark.
Finally, an antifade mounting medium containing DAPI (Solarbio, China) to visualize the nucleus was dripped on the glass slides. The slides were stained for 5 minutes at RT. A fluorescence microscope (IX81, Olympus, Japan) was used to observe and photograph the labeled cells.
Finally, an antifade mounting medium containing DAPI (Solarbio, China) to visualize the nucleus was dripped on the glass slides. The slides were stained for 5 minutes at RT. A fluorescence microscope (IX81, Olympus, Japan) was used to observe and photograph the labeled cells.
6
Immunohistochemical Localization of p65
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The brain slices (4 μm-thick) were laid flat on adhesive glass slides. Initially, the slices were permeabilized with 0.3 % Triton X-100 for 30 min and blocked with goat serum for 1 h. They were then incubated at 4 °C overnight with the primary antibodies, rabbit anti-p65 (1:200, Abcam) followed by incubation with the secondary antibodies, Cy3 anti-rabbit IgG (Cy3-AffiniPure Goat Anti-Rabbit IgG (H + L), 1:200, Jackson, USA) for 2 h at 25 °C. Each step was followed by three times of a 5-min washing in PBS. The prepared specimens were counterstained with DAPI for 10 min and scanned with a Pannoramic MIDI digital slide scanner for image creations (3DHISTECH, Hungary). The localization of p65 expression was analyzed using Caseviewer software (3DHISTECH, Hungary).
7
Chromatin Immunoprecipitation of p65 in Primary Human Monocytes
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Primary human monocytes (2 × 107) were recovered from culture and pelleted prior to crosslinking, performed first in DSG/PBS solution for 30 min at RT, and then in 1% formaldehyde for 10 min at RT, quenched with glycine solution. Dual-crosslinked cells were washed twice in ice cold PBS, pelleted, and stored at −80 °C.
Pellets were thawed on ice and membranes disrupted using a syringe in the presence of protease inhibitors. Chromatin was sheared using a Bioruptor (Diagenode, Denville, NJ). Shearing conditions were optimized for the cell type and number. Sheared chromatin was clarified by centrifugation and immunoprecipitated in duplicate using rabbit anti-p65 (Abcam, Cambridge, UK) or normal rabbit IgG (Cell Signaling Technologies). Chromatin was reserved for normalization (10% of input). Bound chromatin was pulled down using Protein A-agarose beads pre-blocked with salmon sperm DNA (Millipore Sigma) and then de-crosslinked using Chelex 100 and Proteinase K. DNA cleanup was accomplished using a MinElute Kit (Qiagen) according to manufacturer’s instructions. Quantitative PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Primers were designed using the UCSC Human Genome Browser82 (link). Primer sequences may be found in Supplementary Table4 .
Pellets were thawed on ice and membranes disrupted using a syringe in the presence of protease inhibitors. Chromatin was sheared using a Bioruptor (Diagenode, Denville, NJ). Shearing conditions were optimized for the cell type and number. Sheared chromatin was clarified by centrifugation and immunoprecipitated in duplicate using rabbit anti-p65 (Abcam, Cambridge, UK) or normal rabbit IgG (Cell Signaling Technologies). Chromatin was reserved for normalization (10% of input). Bound chromatin was pulled down using Protein A-agarose beads pre-blocked with salmon sperm DNA (Millipore Sigma) and then de-crosslinked using Chelex 100 and Proteinase K. DNA cleanup was accomplished using a MinElute Kit (Qiagen) according to manufacturer’s instructions. Quantitative PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Primers were designed using the UCSC Human Genome Browser82 (link). Primer sequences may be found in Supplementary Table
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