Protein samples of the cells were extracted by lysing the cells with radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The protein samples were quantified using the BCA Protein Assay Kit (Beyotime) and the same amount of samples were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands on the gel were transferred to a polyvinylidene fluoride membrane and blocked in 5% skim milk for 2 h at room temperature and then the blot was incubated in the specific primary antibodies for TSLP (ab47943), skeletal muscle actin (α-SMA, ab5694), collagen I (ab6308), MAPK7 (ab40809), extracellular signal-regulated kinase 1 (ERK1, ab180163), phospho-ERK1 (p-ERK1, ab24157), p38 (ab7952), p-p38 (ab178867), c-Jun N-terminal kinase 1 (JNK1, ab199380), and p-JNK1 (ab47337), purchased from Abcam (Cambridge, UK), overnight at 4°C. β-actin (ab189073) was used as an endogenous reference. After washing, the blot was incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Positive signals were developed using the ECL Plus Western Blotting Substrate (Thermo Scientific). Band densities for each sample were analyzed using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD).
Ab47337
Manufactured by Abcam
Sourced in United States
Ab47337 is a laboratory equipment product offered by Abcam. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab213521, by Abcam (4 mentions)
Ab199380, by Abcam (3 mentions)
Ab6721, by Abcam (2 mentions)
Ab133462, by Abcam (2 mentions)
Ab90463, by Novus Biologicals (2 mentions)
Ab178867, by Abcam (1 mentions)
Ab7952, by Abcam (1 mentions)
Ab47943, by Abcam (1 mentions)
Ab40809, by Abcam (1 mentions)
Ab180163, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
Ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab5694, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Ab24157, by Abcam (1 mentions)
Ab128025, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab133448, by Abcam (1 mentions)
Ab179843, by Abcam (1 mentions)
9 protocols using ab47337
1
Protein Expression Analysis in Cells
2
Immunoblotting Analysis of Autophagy and Apoptosis Markers
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MLO-Y4 cells were homogenized in lysis buffer, and protein concentration was quantified using the Bradford assay. After separation through 15% SDS-PAGE and transferred onto PVDF membranes, cell lysate was subjected to immunoprecipitation with specific antibodies for 1 h at RT, followed by immunoblotting according to the kit’s protocols (Roche, Basel, Switzerland). The primary antibodies used for immunostaining were: rabbit anti-JNK1 (1:1000, Abcam, ab199380), rabbit anti-p-JNK1 ab47337, rabbit anti-AMPK (1:1000, Abcam, ab32382), rabbit anti-p-AMPK (1:1000, Abcam, ab133448), rabbit anti-Beclin 1(1:1000, Abcam, ab207612), rabbit anti-Bcl-2 (1:1000, Abcam, ab182858), rabbit anti-LC3 (1:1000, Abcam, ab128025), rabbit anti-Cleaved Caspase-3 (1:1,000, Abcam, ab32042), rabbit anti-Cleaved PARP (1:1000, Abcam, ab32064), rabbit anti-SOD1 (1:1000, Abcam, ab179843), rabbit anti-SOD2 (1:1000, Abcam, ab74231), rabbit anti-Actin (1:3000, Abcam, ab8227). The secondary antibodies used for immunostaining were: mouse HRP (1:2000, Abcam, ab6728), rabbit HRP (1:2000, Abcam, ab6721).
3
Spinal Cord Protein Expression Analysis
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Spinal cord tissues were homogenized, and total protein was extracted using RIPA lysis, followed by quantitative analysis by bicinchoninic acid (BCA) protein assay kit. 40 μg samples were separated by 10% SDS-PAGE, and then transported to PVDF membranes. After blocking by 5% non-fat milk, the membranes were incubated with primary antibodies against p-IκBα (1:10000, ab133462, Abcam), IκBα (1:800, ab95338, Abcam), p-JNK1(1:1200, ab47337, Abcam), p-JNK2 (1:1000, P02706, Boster), JNK1 (1:2500, ab199380, Abcam), JNK2 (1:1500, 51153-1-AP, Proteintech), β-actin (1:2000, 20536-1-AP, Proteintech), TIPE2 (1:1000, orb158628, Biorbyt), TAK1 (1:800, orb256677, Biorbyt) at 4 ℃ overnight. After continuous incubation with secondary antibody goat anti-rabbit IgG H&L (HRP) (1:3000, ab6721, Abcam) for 1 h, protein bands were observed with enhanced chemiluminescence (ECL) solution. Quantitative result of band was analyzed by Image J 1.49p.
4
ALK Cytotoxicity Evaluation in RAW 264.7 Cells
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Dimethyl sulfoxide (DMSO), ultra-pure E. coli K12, LPS, and MTT were purchased from Sigma. ALK (98% purity) was purchased from Sichuan Victory (ALK structure is shown in Figure 1a ). ELISA kits were purchased from R&D Systems. fetal bovine serum (FBS) and dulbecco’s modified eagle medium (DMEM) were obtained from HyClone. Antibodies were purchased from Abcam (Cambridge, UK) or Proteintech (Rosemont, IL, USA), including anti-iNOS (ab210823, Abcam), anti-COX-2 (ab169782, Abcam), anti-p-P38 (ab195049, Abcam), anti-p38 (ab170099, Abcam), anti-p-ERK1/2 (ab201015, Abcam), anti-ERK1/2 (ab17942, Abcam), anti-p-JNK (ab47337, Abcam), anti-IL-6 (ab233706, Abcam), anti-IL-1β (ab254360, Abcam), anti-TNF-α (ab183218, Abcam), anti-JNK (ab213521, Abcam), anti-p-p65 (ab76302, Abcam), anti-p65 (ab16502, Abcam), anti-p-IκB-α (ab133462, Abcam), anti-IκB-α (ab32518, Abcam), and anti- glyceraldehyde-3-phosphate dehydrogenase (GADPH; ab8245, Abcam).
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ALK had limited cytotoxicity in RAW 264.7 cells. (a) the chemical structure of ALK was presented. (b) RAW264.7 cells were exposed to 1, 3, 5, 10 or 20 μM ALK for 24 h. The cell viability was assessed by MTT assay.
5
Western Blot Analysis of Autophagy Markers
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Proteins were isolated with 12 % SDS–PAGE and then transferred onto homopolymers and copolymer membranes (Schleicher & Schuell, Germany). The membranes were blocked in phosphate-buffered saline (PBS) that containing 10 % nonfat dry milk and 0.5 % Tween-20 overnight. Subsequently, the membranes were incubated with primary antibodies for 2 h. The following antibodies were used: anti- LC3B (Cell Signaling Technology, Danvers, MA, USA; #2775; 1:1000), anti-p62 (Cell Signaling Technology; #5114; 1:1000), anti-HMGB2(Abcam, Cambridge Science Park, UK; ab67282; 1:1000), p-JNK (Abcam, Cambridge Science Park, UK; ab47337; 1:1000), JNK (Abcam, Cambridge Science Park, UK; ab213521; 1:1000), and anti-β-actin (Abcam, Cambridge Science Park, UK; ab8226; 1:1000). The bands were visualized using a chemiluminescence detection system (CWBIO; Beijing, China) and were normalized to that of β-actin.
6
Western Blot Analysis of Protein Markers
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Total protein samples (30 µg) from HMCs were loaded onto the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted on polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). PBS tween-20 (PBST; Sangon Biotech) solution containing 5% skimmed milk was utilized to block the non-specific sites of the membrane. After that, the membrane was labeled with diluted primary antibodies of proliferating cell nuclear antigen (PCNA; ab18197; 1:8000; Abcam, Cambridge, MA, USA), CyclinD1 (ab16663; 1:10,000; Abcam), TGF-β1 (T3176; 1:8000; Sigma), fibronectin (FN; ab2413; 1:5000; Abcam), collagen 4 (Col-4; C1926; 1:5000; Sigma), TIMP3 (ab276134; 1:5000; Abcam), Jun N-terminal Kinase (JNK; ab110724; 1:8000; Abcam), phosphorylated JNK (p-JNK; T183; 1:3000; ab47337) and GAPDH (ab9485; 1:20,000; Abcam). After labeling with horseradish-peroxidase (HRP)-conjugated secondary antibody (1:5000; Abcam), protein bands were visualized using several films and the Super Signal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA). Quantification of protein bands was performed using the Image Lab analysis software (Bio-Rad, Hercules, CA, USA), and the intensities of protein bands were normalized to GAPDH.
7
DRG Protein Expression Analysis
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Briefly, DRG tissues (L4–L6) were removed, and total protein was extracted. The
lysates were centrifuged, and the supernatants were collected. After being
denatured, the supernatant samples containing 20 μg of protein were loaded onto
gels and electrically transferred to a polyvinylidene fluoride membrane. The
membrane was incubated overnight with primary antibodies (diluted at 1:500):
rabbit anti-TRPA1 (1:500, Novus Bio, NB100-91319), anti-TNFR1 (1:500; Abcam
#ab90463), anti-IL-6R (1:500; Abcam#ab103798), anti-p-p38-MAPK (1:500; USBio,
USB#403230)/p38-MAPK (1:500; USBio, USB#403226), anti-p-JNK1(1:500; Abcam
#ab47337)/JNK1 (1:500; Abcam #ab213521). The membranes were washed and incubated
with an alkaline phosphatase conjugated antirabbit secondary antibody (1:1000).
The primary and secondary antibodies were obtained from Abcam Co. or Antibodies
online Com. The immunoreactive proteins were detected by enhanced
chemiluminescence. The bands recognized by the primary antibody were visualized
by exposure of the membrane onto an X-ray film. The membrane was stripped and
incubated with anti-β-actin to show equal loading of the protein. The film was
then scanned, and the optical density of
TRPA1/TNFR1/IL-6R//p-p38-MAPK/p38-MAPK/p-JNK1/JNK1/β-actin bands was analyzed
using the NIH Scion Image software.
lysates were centrifuged, and the supernatants were collected. After being
denatured, the supernatant samples containing 20 μg of protein were loaded onto
gels and electrically transferred to a polyvinylidene fluoride membrane. The
membrane was incubated overnight with primary antibodies (diluted at 1:500):
rabbit anti-TRPA1 (1:500, Novus Bio, NB100-91319), anti-TNFR1 (1:500; Abcam
#ab90463), anti-IL-6R (1:500; Abcam#ab103798), anti-p-p38-MAPK (1:500; USBio,
USB#403230)/p38-MAPK (1:500; USBio, USB#403226), anti-p-JNK1(1:500; Abcam
#ab47337)/JNK1 (1:500; Abcam #ab213521). The membranes were washed and incubated
with an alkaline phosphatase conjugated antirabbit secondary antibody (1:1000).
The primary and secondary antibodies were obtained from Abcam Co. or Antibodies
online Com. The immunoreactive proteins were detected by enhanced
chemiluminescence. The bands recognized by the primary antibody were visualized
by exposure of the membrane onto an X-ray film. The membrane was stripped and
incubated with anti-β-actin to show equal loading of the protein. The film was
then scanned, and the optical density of
TRPA1/TNFR1/IL-6R//p-p38-MAPK/p38-MAPK/p-JNK1/JNK1/β-actin bands was analyzed
using the NIH Scion Image software.
8
Quantification of Neuroinflammatory Proteins in Dorsal Horn
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As described in our previous publication (34 (link)), “the samples containing total protein of the L5-L6 dorsal horn tissues were extracted, centrifuged and the supernatants were collected to assess protein concentrations. Then the supernatant (containing 20 μg of protein) were loaded onto gels and electrically transferred to the membrane. The membrane was incubated overnight with primary rabbit antibodies (1:500), namely anti-TNFR1 (Abcam #ab90463), anti-TRPA1 (Novus Bio, NB100-91319), anti-p-p38-MAPK (USBio, USB#403230) and anti-p-JNK1 (Abcam #ab47337). The primary antibodies were purchased from Abcam Co and/or Antibodies-online Inc. After being washed, the membranes were incubated with anti-rabbit secondary antibody (1:1000, Sigma Co). The bands recognized by immunoreactive proteins were visualized by exposure of the membrane onto an x-ray film. The Scion Image software was employed to determine the optical density of immunoreactive proteins bands.”
9
Western Blot Analysis of DRG Proteins
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Briefly, DRG tissues (L4-L6) were removed and total protein was extracted. The lysates were centrifuged at 15, 000 x g for 15 min and the supernatants were collected. After being denatured, the supernatant samples containing 20 μg of protein were loaded onto gels and electrically transferred to a polyvinylidene fluoride membrane. The membrane was incubated overnight with primary antibodies: rabbit anti-TNFR1 (1:500, Abcam #ab90463), anti-TRPA1 (1:500, Novus Bio, NB100-91319), anti-p-p38-MAPK (1:500, USBio, USB#403230)/ p38-MAPK (1:500, USBio, USB#403226) and anti-p-JNK1(1:500, Abcam #ab47337)/ JNK1 (1:500, Abcam #ab213521). The membranes were washed and incubated (8 hours) with an alkaline phosphatase conjugated anti-rabbit secondary antibody (1:1000, Sigma, Cat#A3687). The immunoreactive proteins were detected by enhanced chemiluminescence. The bands recognized by the primary antibody were visualized by exposure of the membrane onto an x-ray film. The membrane was stripped and incubated with anti-β-actin to show equal loading of the protein. The film was then scanned and the optical density of TRPA1/TNFR1/p-p38-MAPK/ p38-MAPK/p-JNK1/JNK1/β-actin bands was analyzed using the Scion Image software.
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