Western blot analysis was performed as described previously [35 (link)], with slight modifications. Briefly, cells were lysed on ice with radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and sonicated to reduce sample viscosity. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Western blotting was performed using the following antibodies at 4 °C overnight: rabbit anti-β-III-tubulin (1:5000; Covance); rabbit anti-MAP-2 (1:2000; Millipore); rabbit anti-GFAP (1:5000; Abcam); rabbit anti-CNPase, rabbit anti-JNK, rabbit anti-p-JNK, rabbit anti-c-Jun, rabbit anti-p-c-Junser63, and rabbit anti-p-c-Junser73 (all 1:1000; all from Cell Signaling Technology); and mouse anti-β-actin (1:10,000; Sigma-Aldrich). Blots were incubated with horseradish peroxidase-labelled secondary anti-rabbit and anti-mouse antibodies, and immunoreactive bands were visualized on film by enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA) (all 1:10,000, Abcam). Western blots were quantified with ImageJ software from three independent experiments. The intensities of the bands were normalized to β-actin.
Rabbit anti jnk
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-JNK is a primary antibody that specifically recognizes c-Jun N-terminal kinase (JNK) proteins. JNK is a member of the mitogen-activated protein kinase (MAPK) family and plays a role in cellular responses to various stimuli, such as stress, cytokines, and growth factors.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti p38, by Cell Signaling Technology (10 mentions)
Rabbit anti p jnk, by Cell Signaling Technology (9 mentions)
Rabbit anti erk, by Cell Signaling Technology (8 mentions)
Rabbit anti phospho jnk, by Cell Signaling Technology (8 mentions)
Mouse anti β actin, by Merck Group (5 mentions)
Rabbit anti p p38, by Cell Signaling Technology (5 mentions)
Rabbit anti β actin, by Cell Signaling Technology (4 mentions)
Rabbit anti phospho p38, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Rabbit anti p p65, by Cell Signaling Technology (3 mentions)
Rabbit anti p65, by Cell Signaling Technology (3 mentions)
Rabbit anti phospho erk1 2, by Cell Signaling Technology (3 mentions)
Rabbit anti phospho erk, by Cell Signaling Technology (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Rabbit anti erk1 2, by Cell Signaling Technology (2 mentions)
Mouse anti actin, by Merck Group (2 mentions)
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti p erk, by Cell Signaling Technology (2 mentions)
30 protocols using rabbit anti jnk
1
Western Blot Analysis of Neural Markers
2
Western Blotting Analysis of Cardiac Signaling
3
Western Blot Analysis of Inflammatory Markers
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Western blot analyses were performed as previously described [30 (link)]. Protein samples obtained from tissue or BV2 microglia homogenates were separated on 8–12% sodium dodecyl sulfate-polyacrylamide gels, transferred to Immobilon-P membranes (Millipore), and probed overnight at 4 °C with primary antibodies including rabbit anti-sEH (1:1000) and rabbit anti-P65 (1:1000) from Santa Cruz; rabbit anti-iNOS (1:1000) and rabbit anti-cyclooxygenase (COX-2 1:1000) from Cayman; rabbit anti-cCP-3 (1:1000), rabbit anti-p-P38 (1:1000), rabbit anti-P38 (1:2000), rabbit anti-p-C-Jun N-terminal kinase (JNK, Thr183/Tyr185, 1:1000), rabbit anti-JNK (1:2000), rabbit anti-p-extracellular signal-regulated kinase p44/42 (ERK p44/42; Thr202/Tyr204, 1:1000), rabbit anti-ERK (1:2000), and rabbit anti-p-P65(1:1000) from Cell Signaling (Danvers, MA, USA); and mouse anti-β-actin (1:10,000, Sigma-Aldrich). Protein band intensities were quantified using ImageJ software and were normalized to the corresponding β-actin intensity.
4
Protein Expression Analysis in Rat Heart
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The heart tissues (0.1 g) from each group of rats were collected and homogenized in a 1 mL protein extraction buffer. The supernatant was collected after centrifugation, and BCA Protein Assay Kit was used to detect protein concentrations. Total protein samples (20 µg) were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer before subsequent transferal to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were sealed with 5% skimmed milk at 37 °C for 120 min ahead of incubation with the primary antibodies: rabbit β-actin (1:1,000, #4970, Cell Signaling), rabbit anti-cleaved caspase3 (1:1,000, #9662, Cell Signaling), rabbit anti-cleaved caspase9 (1:1,000, #9509, Cell Signaling), rabbit anti-AMPKα1 (1:1,000, #2795, Cell Signaling), rabbit anti-NQO1 (1:1,000, #62262, Cell Signaling), rabbit anti-JNK (1:1,000, #9252, Cell Signaling), rabbit anti-p-JNK (1:1,000, #4668, Cell Signaling), rabbit anti-P65 (1:1,000, #8242, Cell Signaling), rabbit anti-p-P65 (1:1,000, #3033, Cell Signaling) at 4 °C overnight. Subsequently, PVDF membranes were incubated for 60 min at 37 °C with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies. Digital image analysis was conducted with Bio-Rad CFX-96 (Bio-Rad, Hercules, CA, USA) to determine and analyze the density of the bands. β-actin was used as the control.
5
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Beyotime, Shanghai, China) was utilized to obtain the total proteins. After being quantified by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA), equal proteins were loaded and electrophoresed on a polyacrylamide gel, and wet-transferred to PVDF membranes (Millipore, Bedford, MA). Then the membranes were kept in 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) at indoor temperature for 1.5 h and kept in primary antibody solution with a 1:1000 dilution ratio at 4 °C for 12 h. After being treated with secondary antibody solution with a 1:5000 dilution ratio for 1 h at ambient temperature, the membranes were observed with ECL Plus WB detection reagents (Millipore).
The primary antibodies including mouse anti-phosphorylated (p)-p38, mouse anti-p38, rabbit anti-p-extracellular regulated MAP kinase (p-ERK), rabbit anti-ERK, rabbit anti-p-c-Jun NH2-terminal (p-JNK), rabbit anti-JNK, rabbit anti-β-actin, rabbit anti-p-Syk, rabbit anti-Syk, rabbit anti-p- protein kinase C delta (PKC δ), rabbit anti-PKC δ, rabbit anti-p-IKK α/β, rabbit anti-IKK α, rabbit anti-p-P65, and rabbit anti-P65 were bought from Cell Signaling Technology (Danvers, Massachusetts, USA). The rabbit anti-Histone 3 antibody was bought from Abcam (Cambridge, Massachusetts, USA).
The primary antibodies including mouse anti-phosphorylated (p)-p38, mouse anti-p38, rabbit anti-p-extracellular regulated MAP kinase (p-ERK), rabbit anti-ERK, rabbit anti-p-c-Jun NH2-terminal (p-JNK), rabbit anti-JNK, rabbit anti-β-actin, rabbit anti-p-Syk, rabbit anti-Syk, rabbit anti-p- protein kinase C delta (PKC δ), rabbit anti-PKC δ, rabbit anti-p-IKK α/β, rabbit anti-IKK α, rabbit anti-p-P65, and rabbit anti-P65 were bought from Cell Signaling Technology (Danvers, Massachusetts, USA). The rabbit anti-Histone 3 antibody was bought from Abcam (Cambridge, Massachusetts, USA).
6
Western Blot for Protein Analysis
7
Protein Expression and Oxidative Stress
8
Western Blot Analysis of Apoptosis Markers
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Western blots were performed as described [38] (link). Mouse monoclonal anti-Caspase 8 (Sigma), mouse monoclonal anti-Caspase 9 (Sigma), rabbit anti-Caspase 3 (Sigma), rat monoclonal anti-Caspase 12 (Sigma), rabbit polyclonal antibody against Bim (amino acids 4–195 of BimEL form), rabbit anti-Cytochrome C (Cell Signaling), goat polyconal anti-HP1 (Santa Cruz), rabbit anti-CoxIV (Cell Signaling), rabbit anti-phospho-JNK (Thr183/Tyr185) (81E11) (Cell Signaling), rabbit anti-JNK (Cell Signaling), mouse monoclonal anti-CHOP (L63F7) (Cell Signaling), rabbit anti-Apaf-1 (Cell Signaling), rabbit anti-Bcl2 (Cell Signaling), rabbit polyclonal anti-ATF-6 (Santa Cruz), rabbit anti-Bax (Cell Signaling) were all used at a 1:1000 dilution for western blots.
9
Inflammatory Pathway Modulation Protocols
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Amlexanox was purchased from Tokyo Chemical Industry (Tokyo, Japan). STAT3 activation inhibitor SPI was purchased from BioVision (Milpitas, CA, USA). LPS from Escherichia coli O111:B4 was obtained from Sigma–Aldrich (St. Louis, MO, USA). The following primary antibodies, which were diluted at 1:1000 ratios in EveryBlot Blocking Buffer (Bio-Rad Laboratories), were used for Western blotting (WB): rabbit anti-COX2 (Cell Signaling Technology), mouse anti-iNOS (Cell Signaling Technology), rabbit anti-p-AKT (Ser473, Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), rabbit anti- IκBα (Cell Signaling Technology), rabbit anti-p- IκBα (Ser32, Cell Signaling Technology), rabbit anti-IKKε (Cell Signaling Technology), rabbit anti-ERK (Cell Signaling Technology), rabbit anti-p-ERK (Thr202/Tyr204, Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-p-STAT3 (Tyr705, Cell Signaling Technology), rabbit anti-NF-κB p65 (Cell Signaling Technology), rabbit anti-p-NF-κB p65 (Ser536, Cell Signaling Technology), rabbit anti-JNK, rabbit anti-p-JNK (Thr183/Tyr185, Cell Signaling Technology), rabbit anti-p38 (Cell Signaling Technology), and rabbit anti-p-p38 (Thr180/Tyr182, Cell Signaling Technology), and mouse anti-β-actin (Santa Cruz Biotechnology). Other chemicals for Western blotting were obtained from Bio-Rad Laboratories.
10
Western Blot Analysis of Protein Signaling
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After treatment, protein extracts for total cellular fractions were isolated in a cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) supplemented with 0.1 mg PMSF and a 1/100 dilution of protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Scraped samples were then centrifuged at 10000 rpm for 15 min at 4°C. The supernatants were used for Western blotting. Protein concentrations were measured using the Bio-Rad protein dye microassay (Bio-Rad, Hercules, USA). Proteins were separated on a 12% SDS-PAGE gel and then transferred onto PVDF membranes. The membrane was blocked with a solution of TBS and 5% fat-free milk for 1 h, then incubated overnight with rabbit anti-A-FABP, rabbit anti-phospho-JNK, rabbit anti-JNK, rabbit anti-TLR4 (all from Cell Signaling Technology, Beverly, MA, USA), or monoclonal mouse anti-β-actin (Santa Cruz Biotechnology, Heidelberg, Germany). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG in TBS for 2 h at room temperature. The membrane was then exposed to film before development.
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