Tumors sections were obtained from NPC xenograph mice and were fixed with 10% formaldehyde at 4°C for 8 h and embedded in paraffin. Tumor samples were cut into sections (6 mm) and antigen retrieval was also performed by incubating tissue sections at 93°C for 20 min, followed by incubation on ice for 40 min. Dehydration was performed by passing a graded concentrations of ethanol (100, 95, 85, 70 and 50%) Tumor sections were incubated with rabbit anti-human JNK (1:1,200; ab112501), caspase-3 (1:1,200; ab13847), hypoxia-inducible factor (HIF)1α (1:1,200; ab51608), tumor necrosis factor (TNF)-α (1:1,200; ab6671) and VEGF (1:1,200; ab46154; all Abcam) antibodies for 2 h at 37°C, followed by incubation with anti-rabbit IgG-HRP secondary antibody (1:1,000; MBS435036; MyBioSource, San Diego, CA, USA) at room temperature for 1 h and washing with PBS. VENTANA System Software 12.3 (BenchMark, San Francisco, CA, USA) was used for observation of proteins.
Ab112501
Manufactured by Abcam
Sourced in United States
Ab112501 is a lab equipment product. It is a core product that serves a specific function. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab4821, by Abcam (4 mentions)
Ab170099, by Abcam (3 mentions)
Ab17942, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab6671, by Abcam (1 mentions)
Ab51608, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab46154, by Abcam (1 mentions)
Mbs435036, by MyBioSource (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Sc 8019, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
P stat3, by Santa Cruz Biotechnology (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
P jnk, by Abcam (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Ab54230, by Abcam (1 mentions)
Ab215206, by Abcam (1 mentions)
Ab169755, by Abcam (1 mentions)
Ab21981, by Abcam (1 mentions)
10 protocols using ab112501
1
Immunohistochemical Analysis of Tumor Proteins
2
Analyzing IL-6-induced Transcription Factor Phosphorylation
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To analyze the IL-6-induced phosphorylation of transcription factors, LF fibroblasts grown in a 6-well plate were incubated for 15 min with IL-6 (300 ng/ml) and lysed in radioimmmunoprecipitation assay buffer (Nacalai Tesque). The lysates were homogenized in sample buffer solution containing 2-mercaptoethanol (2×) (Nacalai Tesque) and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Invitrogen) that was blocked in blocking reagent (Block-Ace; DS Pharma Biomedical, Osaka, Japan) for 30 min at 25 °C and treated overnight at 4 °C with the following primary antibodies: anti-human STAT3 (mouse monoclonal, 1:500, SC-8019) and p-STAT3 (1:500) (both from Santa Cruz Biotechnology); and anti-human ERK1/2 (mouse monoclonal, 1:1000, AB54230), p-ERK1/2 (1:1000), p38 (rabbit monoclonal, 1:1000, AB170099), p-p38 (1:1000), JNK (rabbit polyclonal, 1:500, AB112501), and p-JNK (1:500) (all from Abcam). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at 25 °C and immunoreactivity was visualized with Chemi-Lumi One Super (Nacalai Tesque) and a luminescent image analyzer (EZ-capture2; ATTO, Tokyo, Japan) and quantified using ImageJ v.1.47 software (National Institutes of Health, Bethesda, MD, USA).
3
Protein Isolation and Analysis in Lung Tissue
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Total proteins were isolated from right lung tissue (50 µg) and cells (5×106) using RIPA buffer (#89900; Thermo Scientific, Worcester, MA, USA) containing a protease inhibitor cocktail (#P2714; Sigma) and phosphatase inhibitor cocktail (#04906845001; Roche Applied Science, Indianapolis, IN, USA). Homogenates were centrifuged at 2,834 g at 4 °C for 20 min before the supernatant was collected. The total protein concentrations were determined by bicinchoninic acid assay (#23227, PierceTM Biotechnology, Rockford, IL, USA). Standard SDS-PAGE technique (27 (link)) was performed on equal amounts of proteins, with antibodies of p-STAT5 (ab32364; 1:500), RORγt (ab135669; 1:500), FOXP3 (ab215206; 1:500), JNK (ab112501; 1:500), p-JNK (ab4821; 1:500), arachidonate 5-lipoxygenase (Alox5; ab169755; 1:500), activator protein 1 (AP-1, ab21981; 1:500), and GAPDH (ab181602; 1:500) obtained from Abcam. The band intensity was measured by ImageJ2× 2.1.4.7 (Wayne Rasband, National Institutes of Health, USA).
4
Western Blot Analysis of MAPK Signaling in Hippocampal Tissue
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Brain tissue from the hippocampal region was extracted promptly after anesthesia at each specific time point after BCCAO surgery. Protein content was detected by applying BCA kit (Beyotime, Shanghai, China). SDS-PAGE gels were used to load and separate protein samples, which were then transferred to polyvinylidene difluoride membranes (Millipore, Inc., USA). After blocking in 5% skim milk for 2 h, membranes were incubated overnight with the following primary antibodies: anti-ERK1/2 rabbit antibody (no. 4695, CST, USA, 1:1,000), anti-p-ERK1/2 rabbit antibody (no. 4370, CST, USA, 1:2,000), anti-p38MAPK rabbit antibody (no. 8690, CST, USA, 1:1,000), anti-p-p38MAPK rabbit antibody (no. 4511, CST, USA, 1:1,000), anti-GADPH mouse antibody (no. 97166, CST, USA, 1:5,000), anti-JNK1/2 (no.ab112501, Abcam, USA, 1:1,000), anti-p-JNK1/2 (no.ab4821, Abcam, USA, 1:1,000). The secondary antibody was then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C. The membranes were examined using a gel imaging device (Vilber Lourmat fusion FX 7 Spectra, France) and the results were analyzed using software (FUSION-CAPT, France).
5
Western Blot Analysis of Key Proteins
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Western blot analysis was performed as described in our previous report [13 (link), 14 (link)]. These primary antibodies are as followed:β-actin (ab8227) (1:3,000), CASC3 (ab90651), p38 (ab31828), phospho p38 (ab47363), ERK1/2 (ab17942), phospho ERK1/2 (ab50011), JNK1/2 (ab112501), and phospho JNK1/2 (ab131499) (1:1,000; ABcam, Cambridge, MA, USA).
6
Western Blotting Analysis of MAPK Signaling
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Protein was extracted using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The protein samples were resolved by 10% SDS-PAGE and were transferred to the PVDF membrane. Next, the membranes were blocked with 5% skim milk at 25 °C for 1 h, then were incubated with the primary antibodies AEBP1 (1:1000, ab168355, Abcam), JNK1/2 (1:1000, ab112501, Abcam), p-JNK1/2 (1:1000, ab4821, Abcam), p38 (ab170099, Abcam), p-p38 (1:1000, ab178867, Abcam) and GAPDH (ab9485, Abcam) overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 25 °C. Blots were detected by enhanced chemiluminescence reagent and western blotting reagents. GAPDH was employed as a protein loading control.
7
Crocin and Inflammatory Pathways
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Crocin (purity assay of ≥ 98%) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. Methylthiazolyldiphenyl-tetrazolium bromide (MTT), LPS, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), fluorescein isothiocyanate (FITC)-albumin, and dimethyl-sulfoxide (DMSO) were obtained from Sigma-Aldrich. Rabbit monoclonal antibody to HMGB1 (ab79823), NF-κB p65 (ab32536), IκBα (ab32518), p-IκBα (ab133462), JNK (ab112501), ERK (ab184699), p38 (ab170099), p-ERK (ab201015), p-JNK (ab4821), p-p38 (ab47363), LaminB1 (ab133741), MMP-9 (ab38898), HPA (ab85543), SDC-4 (ab24511), Ly6g (ab25377), β-actin (ab8224), GAPDH (ab181602), MMP-9 inhibitor (MMP-9 inhib, ab142180), and FITC (ab25539) were acquired from Abcam Trading Company Ltd. Mouse polyclonal antibody to cathepsin L (CTL) was purchased from Santa Cruz Biotechnology. Mouse polyclonal antibody to HS was purchased from AMS Biotechnology (Switzerland, MA). Thrombomodulin/BDCA-3 was obtained from R&D Systems (USA). HRP-conjugated goat anti-rabbit IgG, goat anti-rabbit IgG/Alexa Fluor 594, and rabbit anti-goat IgG/Alexa Fluor were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. Cathepsin L inhibitor (CTL inhib, CAS 167498-29-5) was obtained from Santa Cruz Biotechnology.
8
Protein Expression Analysis Protocol
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RIPA buffer containing 1% protease inhibitor was added to cells or tissues, which were subsequently lysed on ice for 30 mins. The lysed cells or tissues were then centrifuged for 20 mins, and the supernatants were collected. The total protein concentration in each supernatant was determined by the BCA method. Next, a 30 μg aliquot of protein from each sample was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Millipore, Burlington, MA, USA), which were subsequently blocked with 5% skim milk for 1.5 hrs. Next, the membranes were washed and then incubated overnight at 4°C with the following primary antibodies (1∶1000, Abcam, Cambridge, UK): IRAK1 (Abcam, ab238), E-cadherin (Abcam, ab15148), N-cadherin (Abcam, ab18203), vimentin (Abcam, ab137321), IL-1β (Abcam, ab9722), NLRP3 (Abcam, ab214185), ASC (Abcam, ab227502), ERK1/2 (Abcam, ab17942), JNK1/2 (Abcam, ab112501), p-ERK1/2 (Abcam, ab223500), p-JNK1/2 (Abcam, ab4821), Caspase 3 (Abcam, ab32042), and GAPDH (Abcam, ab9485). The next day, the membranes were washed and then incubated with a 1∶5000 diluted secondary antibody (Abcam) for 2 hrs. The immunostained proteins were detected using an ECL substrate kit (Thermo Scientific, Waltham MA, USA), and results were displayed on a Bio-Rad Gel Doc/Chemi Doc Imaging System.
9
Immunochemical Marker Detection in Neurodegenerative Research
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Rabbit IBA1 (234 004, 1:500 for IHC; Synaptic Systems), PLIN2 (GP40, 1:500 for histology, 1:3,000 for Western blotting; PROGEN), p-eIF2α (ab32157, 1:200 for staining, 1:2,000 for Western blotting; Abcam), total eIF2α (2103, 1:2,000 for Western blotting; Cell Signaling Technology), JNK1+JNK2 (p-T183+Y185, ab4821, 1:1,000 for Western blotting; Abcam), JNK1+JNK2 (ab112501, 1:1,000 for Western blotting; Abcam), LAMP1 (sc-19992, 1:100 for histology; Santa Cruz Biotechnology), TREM2 (AF1729, 1:200 for histology; R&D Systems), α-tubulin clone B-5-1-2 (T5168; Sigma-Aldrich); GAPDH (ab9484, 1:5,000 for Western blotting; Abcam), GFAP (173004, 1:500; Synaptic Systems), MAC2 (125402, 1:250; BioLegend), APC (OP80, 1:100; Merck Calbiochem), and oligodendrocyte transcription factor 2 (OLIG2; ab9610, 1:200; Millipore).
10
Western Blot Analysis of Proteins
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After infecting lentivirus, the cells in in logarithmic growth status were collected to extract total proteins. Twenty microgram proteins were segregated by 12% SDS-PAGE and transferred into PVDF membrane for western blot analysis. The PVDF membrane was blocked with a blocking solution (1 × TBST solution containing 5% skimmed milk) at room temperature for 1 h. Then, the primary antibody was added and incubated at room temperature for 2 h or overnight at 4 °C, and then washed with 1 × TBST solution for 3 times, 10 min each time. Thereafter, the corresponding secondary antibody was added. Finally, the chemiluminescence was evaluated by using a chemiluminescence imager.
The primary antibodies used in western blotting were as follows: RPL35A (1:2000, Abcam, #ab241070), GAPDH (1:3000, Bioworld, #60004-1-lg), JNK1 + JNK2 (1:500, Abcam, #ab112501), p-JNK1 + JNK2 (1:500, Abcam, #ab131499), P38 (1:1000, CST, #8690S), p-P38 (1:500, CST, #bs-5476R) and p53 (1:5000, proteintech, #60283-2-Ig). The secondary antibodies used in western blotting were Goat Anti-Rabbit (1:3000, Beyotime, #A0208) and Goat Anti-Mouse (1:3000, Beyotime, #A0216).
The primary antibodies used in western blotting were as follows: RPL35A (1:2000, Abcam, #ab241070), GAPDH (1:3000, Bioworld, #60004-1-lg), JNK1 + JNK2 (1:500, Abcam, #ab112501), p-JNK1 + JNK2 (1:500, Abcam, #ab131499), P38 (1:1000, CST, #8690S), p-P38 (1:500, CST, #bs-5476R) and p53 (1:5000, proteintech, #60283-2-Ig). The secondary antibodies used in western blotting were Goat Anti-Rabbit (1:3000, Beyotime, #A0208) and Goat Anti-Mouse (1:3000, Beyotime, #A0216).
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