IPFP-MSCs or OACs were fixed in 4% paraformaldehyde. Non-specific bindings were blocked with QuickBlock blocking buffer (Beyotime) for 2 h. Fixed cells were incubated with primary antibodies (collagen II, aggrecan, SOX-9, VCL, integrin β1, ERK1/2 and p-ERK1/2) (Abcam) overnight at 4 °C, followed by incubation with secondary antibody (Ms-647 or Rb-488) (Abcam) for 90 min at room temperature. Nuclei were stained with DAPI (Beyotime) for 8 min. Samples were observed by Laser scanning confocal microscope (LSM780 ZEISS). The relative fluorescence unit (RFU) was analyzed using ZEN 2012 software. Three different fluorescence images among each group were used for statistical analysis (total fluorescence intensity of the image/number of nuclei).
P erk1 2
Manufactured by Abcam
Sourced in United Kingdom, United States, China
P-ERK1/2 is a primary antibody that detects phosphorylated forms of the extracellular signal-regulated kinase (ERK) 1 and 2 proteins. ERK1/2 are serine/threonine protein kinases that play a crucial role in the regulation of cell growth, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Abcam (38 mentions)
β actin, by Abcam (10 mentions)
P jnk, by Abcam (10 mentions)
P akt, by Abcam (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Gapdh, by Abcam (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
E cadherin, by Abcam (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Mmp 9, by Abcam (4 mentions)
P akt, by Cell Signaling Technology (4 mentions)
P mek1 2, by Abcam (4 mentions)
Bcl 2, by Abcam (4 mentions)
Bcl2 associated x (bax), by Abcam (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Proteintech (3 mentions)
Mek1 2, by Abcam (3 mentions)
P jnk, by Cell Signaling Technology (3 mentions)
Gsk 3β, by Abcam (3 mentions)
78 protocols using p erk1 2
1
Immunofluorescence Analysis of MSCs and OACs
2
Immunostaining of Brain Tissue Sections
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If appropriate, for brain tissues, four-micron frozen brain sections that were fixed in 4% formaldehyde or acetone were blocked for endogenous peroxidase activity with 0.03% H2O2. PHNs were cultured on glass coverslips that were precoated with poly-L-lysine (Sigma-Aldrich) and then were fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 at room temperature for 10 min. Blocking was achieved with PBS that contained 5% normal goat serum at room temperature for 1 h.
Sections were then incubated at 4°C overnight with the following primary antibodies: IGF-I (monoclonal, 1:200), pERK1/2 (monoclonal, 1:400), pnNOS Ser1417 (monoclonal, 1:200), MAP2 (monoclonal, 1:500; Abcam), pRSK (monoclonal, 1:400; Biorbyt). Binding of primary antibodies was revealed by incubating the sections for 30 min with Alexa Fluor 488 (green)/Alexa Fluor 594 (red)-conjugated secondary antibodies (Abcam) or with horseradish peroxidase-labeled secondary antibodies (Abcam). They were then visualized by diaminobenzidine.
Sections were then incubated at 4°C overnight with the following primary antibodies: IGF-I (monoclonal, 1:200), pERK1/2 (monoclonal, 1:400), pnNOS Ser1417 (monoclonal, 1:200), MAP2 (monoclonal, 1:500; Abcam), pRSK (monoclonal, 1:400; Biorbyt). Binding of primary antibodies was revealed by incubating the sections for 30 min with Alexa Fluor 488 (green)/Alexa Fluor 594 (red)-conjugated secondary antibodies (Abcam) or with horseradish peroxidase-labeled secondary antibodies (Abcam). They were then visualized by diaminobenzidine.
3
PKCδ-Targeting siRNA and Tanzisertib Protocol
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The PKCδ-specific siRNA and negative-control siRNA were designed and synthesized by GenePharma (Shanghai, China), and LipofectamineTM 3000 Reagent was purchased from Thermo Fisher. Tanzisertib (CC-930) was purchased from Sellectchem (Sellectchem, Houston, TX, USA) and dissolved in DMSO. Primary antibodies against c-Myc, CDK2, CDK6, Cyclin D2, P21cip1, P27kip1, PCNA, cytochrome C, PKCδ, PKCα, PKCη, PKCβ, PKCζ, IL-6, ERK1/2, p-ERK1/2, P38/MAPK, p-P38/MAPK and GSDME were obtained from Abcam (Cambridge, UK). Antibodies against Caspase-3, Caspase-8, cleaved Caspase-8, Caspase-9, cleaved Caspase-9, PARP, Bax, Bcl-2, Cyclin D1, Survivin, SAPK/JNK, p-SAPK/JNK, NF-κB/P65, p-NF-κB/P65, Caspase-1 and NLRP3, along with anti-mouse and anti-rabbit secondary antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA). FastStart Universal SYBR Green Master (Rox) was purchased from Sigma-Aldrich (Roche, Germany). Primers were prepared by Invitrogen (Shanghai, China). Other reagents were analytical-grade or guaranteed reagent commercial products, and were used without further purification, unless otherwise noted.
4
Western Blot Analysis of Protein Expression
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RIPA buffer containing proteinase inhibitors (Beyotime, China) was used to create tissue extracts, which were then used to perform the analyses., and the concentration of protein was measured by BCA Protein Assay Reagent (Beyotime, China). The tissue lysate containing 40μg of total protein was loaded onto the SDS-PAGE gel and electrophoresed at 120V for 90 minutes, and then the protein to be tested in the gel was transferred to a 0.45m polyvinylidene fluoride membrane (Merck Millipore, Germany). After blocking the membrane in TBST buffer containing 5% skimmed milk powder for 30 minutes, the membranes were incubated for 12 hours at 4 °C with the following antibodies: CXCR4 (Abcam, UK), GAPDH (Proteintech, China), β-tubulin (Proteintech, China), ERK1/2 (Abcam, UK), p-ERK1/2 (Abcam, UK). The membranes were rinsed three times with TBST (5 minutes/wash) the next day before being incubated with goat anti-rabbit secondary antibody (Thermo Fisher, USA) or goat anti-mouse secondary antibody (Thermo Fisher, USA) in 5% skimmed milk powder /TBST, and then rinsed 3 times. Membrane development was carried out using ECL Substrate (Thermo Fisher, USA), and the chemiluminescence was captured using the bioanalytical imaging system C300 (Azure Biosystems, USA) and analyzed using ImageJ software.
5
Investigating Fibrosis Regulation Mechanisms
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BLM was manufactured by Nippon Kayaku Co., Ltd (batch number: 640412). Prednisone acetate was purchased from Guangdong South China Pharmaceutical Group Co., Ltd (batch number: 140704). Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St. Louis, MO, United States, batch number: 025M4040Y). Recombinant mouse TGF-β1 was purchased from PeproTech (No. 218, Xinghu St, SIP Suzhuo, China) and used at a concentration of 5 ng/ml. N-acetylcysteine was produced by MedChemExpress LLC (Princeton, NJ, United States of America, batch number: HY-B0215/CS-2160). Primary antibodies against TGF-β1, α-SMA, Smad3, Smad4, p-Smad3, p-Smad4, Smad7, CTGF, ERK1/2, p-ERK1/2, β-actin, and GAPDH were purchased from Abcam (Cambridge, United Kingdom).
6
Western Blot Analysis of Brain Proteins
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Brain tissues were lysed with RIPA buffer containing a cocktail of protease inhibitors and phosphatase inhibitors. Lysate was centrifuged at 14,000 rpm at 4 °C for 15 min and the supernatant was collected. The total protein concentration was determined by using the Bradford assay (Bio-rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded on 10% SDS-PAGE gel and then transferred onto a PVDF membrane (Millipore, Temecula, CA, USA). After blocking with 5% skim milk or 3% BSA, the membrane was incubated overnight at 4 °C with primary antibodies against BDNF, TrkB, PSD-95, Erk1/2, pErk1/2 (Abcam, Cambridge, UK), and GAP-43 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS-T, the membrane was incubated with the proper secondary antibody conjugated to HRP for 2 h at room temperature. Interested protein signals on the membrane were detected by using SuperSignal West Pico chemiluminescence substrate (Thermo Fisher Scientific, Rockford, IL, USA). The signal intensity of each band on the film was quantified by using ImageJ software (Java 8) and normalized to the corresponding β-actin (Cell Signaling Technology, Danvers, MA, USA) as an internal control.
7
Validating Brain Protein Levels in ICH
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Validation of LC-MS/MS results of selected proteins in brain homogenates from the sham and ICH rats was performed by means of western blot. Briefly, equal protein amounts (from 50 to 80 μg depending on each specific protein) of brain homogenates were separated on 12.5% polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and then transferred to a PVDF membrane (Millipore). Membranes were blocked for 1 h in 5% skim milk within TBS-T buffer and incubated overnight at 4 °C with the following antibodies: rabbit albumin (1:3000, Abcam, USA), rabbit ERK1/2 (CST1:1000, Abcam, USA), P-ERK1/2 (CST1:2000, Abcam, USA), primary GAPDH antibody (1:3000, Abcam, USA), and the secondary antibody (goat-anti-rabbit IgG conjugated to horseradish peroxidase (1:5000, Abcam, USA)). The specific reaction was visualized by chemiluminescence of the substrate luminal reagent (GE Healthcare, UK). Blot images were then scanned. The optical density of protein levels was quantified using IPP software (version 5.1, Media Lybernetics, USA), corrected for protein load determined by GAPDH, and expressed as the relative value to the controls. Three rats were used in each group.
8
Immunoblotting Analysis of BM-DCs Activation
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Mature BM-DCs were placed in six-well plates with a density of 1.5 × 106 cells/ml and stimulated with 100 ng/ml LPS to induce DC activation. Meanwhile, 100 μM Scopoletin was added to DCs. After 18 h, proteins were extracted from BM-DCs as described. The total protein contents were measured by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, MA). DC proteins were equally loaded onto SDS-PAGE, electrophoresed, transferred onto PVDF membranes, and after blocking with 5% nonfat milk in TBS, the PVDF membranes were incubated with primary antibody over 12 h at 4°C (Li et al., 2018 (link)), including JNK, p-JNK, NF-κB, p-NF-κB, p38, p-p38 (cell signal technology), IRF-7, ERK1/2, p-ERK1/2 (Abcam, Cambridge, UK), and p-IRF-7 (Signalway Antibody, College Park, MD). After washing, the membranes were incubated with 0.5% horseradish peroxidase-labeled IgG. Temembrane-bound antibodies were detected with an Immolilon™ Western chemiluminescent HRP Substrate (Millipore, Billerica, MA) and analyzed with an ImageJ (ChemiDoc XRS System; Bio-Rad).
9
Western Blot Analysis of Protein Signaling
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Protein extracts (30 µg per lane) were electrophoresed and then transferred to polyvinylidene fluoride membrane. Immuno-blotting was performed by incubating each membrane with an anti-HGF (dilution: 1∶1000; Abcam, Cambridge, UK), c-MET (dilution: 1∶250; Abcam, Cambridge, UK), AKT (dilution: 1∶1000; Abcam, Cambridge, UK), p-AKT (dilution: 1∶1000; Abcam, Cambridge, UK), ERK1/2 (dilution: 1∶1000; Abcam, Cambridge, UK), p-ERK1/2 (dilution: 1∶1000; Abcam, Cambridge, UK), MMP-2 (dilution: 1∶1000; Abcam, Cambridge, UK), MMP-9 (dilution: 1∶500; Abcam, Cambridge, UK), cyclin D1 (dilution: 1∶250; Abcam, Cambridge, UK) or β-actin overnight at 4°C. After being washed in PBS, each membrane was incubated for 1 h with a secondary antibody conjugated by peroxidase at room temperature. The band was developed by use of enhanced chemi-luminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The density of each band was determined. The results were repeated twice to confirm the reproducibility.
10
Protein Expression Analysis by Western Blot
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Protein extracts were boiled for 5 min in SDS sample buffer, separated using SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Immobilon™-P, MerckMillipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies: rabbit anti-MMP-2 (1:1000; Novus Biologicals, Littleton, CO, USA), mouse anti-MMP-9 (1:300; Abcam, Tokyo, Japan), Akt (1:1000; Santa Cruz, California, USA), pAkt (1:1000; Santa Cruz), p65 NF-κB (1:1000; Santa Cruz), IкB-α (1:1000; Santa Cruz), ERK1/2 (1:1000; Abcam, Cambridge, UK), pERK1/2 (1:1000; Abcam), p38 (1:1000; Abcam), p-p38 (1:1000; Abcam), JNK (1:1000; Abcam), pJNK (1:1000; Abcam), c-Fos (1:500; Santa Cruz), c-Jun (1:500; Santa Cruz), iNOS (1:1000; Santa Cruz), and mouse anti-β-actin (1:10,000; Merck). All immunoreactive proteins were detected using ECL™ Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).
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