Total cell or tissue extracts were extracted by cell lysis buffer followed by immunoblotting with anti-Ras (1:2,000; Proteintech), anti-β-actin (1:5,000; Proteintech), anti-ERK (1:1,000; Proteintech), anti-pERK (1:500; Proteintech), anti-MEK (1:1,000; Proteintech), and anti-pMEK (1:200; Proteintech). 25 µg of cell lysates was resolved with 12.5% SDS–PAGE and transferred to polyvinylidene fluoride. After 1-h blocking with blocking buffer, the membranes were incubated with primary antibody for 2 h at room temperature. After washing and 1-h incubation with secondary antibodies at room temperature, the blots were visualized using ECL.
Anti erk
Manufactured by Proteintech
Sourced in United States, China
Anti-ERK is a laboratory reagent that binds to and detects the presence of the extracellular signal-regulated kinase (ERK) protein. It is used as a tool in biochemical and cell biology research applications.
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Lab products found in correlation
Anti perk, by Proteintech (7 mentions)
Anti β actin, by Proteintech (5 mentions)
Anti gapdh, by Proteintech (4 mentions)
Anti p erk, by Cell Signaling Technology (3 mentions)
Anti akt, by Proteintech (3 mentions)
Anti bcl 2, by Proteintech (3 mentions)
Anti bax, by Proteintech (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti ras, by Proteintech (2 mentions)
Anti mek, by Proteintech (2 mentions)
Anti pmek, by Proteintech (2 mentions)
Anti mouse, by Jackson ImmunoResearch (2 mentions)
Anti rabbit, by Jackson ImmunoResearch (2 mentions)
Ag 20b 0042, by Abcam (2 mentions)
Anti piκb, by Proteintech (2 mentions)
Anti iκb, by Proteintech (2 mentions)
Anti gapdh, by Jackson ImmunoResearch (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Anti cyclin d1, by Proteintech (2 mentions)
Anti mcl1, by Proteintech (2 mentions)
15 protocols using anti erk
1
Immunoblotting for Ras, ERK, and MEK
2
OXGR1 Signaling Pathway Analysis
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HEK293 cells were transiently transfected with plasmids expressing empty vector or FLAG-tagged OXGR1 using PolyJet reagent. The medium was switched to serum-free DMEM 36 hours after transfection. After serum starvation for another 12 hours, cells were stimulated with the indicated ligand and lysed in Laemmli buffer. Protein lysate was separated on standard SDS-PAGE, transferred onto a PVDF membrane, and then subjected to immunoblotting with the following antibodies: anti–p-ERK (catalog 4370, Cell Signaling Technology); anti-Flag (catalog 14793, Cell Signaling Technology); anti-IRG1/anti-ACOD1 (catalog 19857, Cell Signaling Technology); anti-Myc (catalog 2278, Cell Signaling Technology); anti-ERK (catalog 67170-1-Ig, Proteintech); and anti-actin (catalog A00702, GenScript).
3
Western Blot Analysis of Inflammasome
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For caspase-1 analysis, BMDMs were lysed together with the supernatant using 50 μl caspase lysis buffer (1× protease inhibitors, 1× phosphatase inhibitors, 10% NP-40, and 25 mM DTT). Then 125 μl 4× SDS loading buffer was added for boiling the samples. For analysis of all other proteins, the supernatants of stimulated cells were discarded, and then cells were washed once with PBS, after which RIPA lysis buffer was added to lyse the cells.
Electrophoresis was conducted to separate proteins in 10%−12% polyacrylamide gels. Then proteins were transferred onto PVDF membranes and blocked with 5% skim milk for 1 h at room temperature. All the primary antibodies were incubated overnight at 4°C, while secondary antibodies with HRP were incubated for 1 h at room temperature. Images were developed via a GE Amersham Imager 600. And final images were analyzed via Fiji for MacOS X (version 2.0.0-rc-67/1.52c).
The antibodies used were: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:2000), anti-GSDMD (abcam, ab209845, 1:1000), anti-pERK (Cell Signaling Technology [CST], #9101, 1:1000), anti-pIκB (CST, #2859, 1:1000), anti-ERK (CST, #9102, 1:1000), anti-IκB (CST, #9242, 1:1000), anti-β-actin (Proteintech, 66009–1-IG, 1:5000), anti-GAPDH (CST, #5174, 1:1000) and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, anti-rabbit [111–035-047], 1:5000; anti-mouse [315–035-047], 1:5000).
Electrophoresis was conducted to separate proteins in 10%−12% polyacrylamide gels. Then proteins were transferred onto PVDF membranes and blocked with 5% skim milk for 1 h at room temperature. All the primary antibodies were incubated overnight at 4°C, while secondary antibodies with HRP were incubated for 1 h at room temperature. Images were developed via a GE Amersham Imager 600. And final images were analyzed via Fiji for MacOS X (version 2.0.0-rc-67/1.52c).
The antibodies used were: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:2000), anti-GSDMD (abcam, ab209845, 1:1000), anti-pERK (Cell Signaling Technology [CST], #9101, 1:1000), anti-pIκB (CST, #2859, 1:1000), anti-ERK (CST, #9102, 1:1000), anti-IκB (CST, #9242, 1:1000), anti-β-actin (Proteintech, 66009–1-IG, 1:5000), anti-GAPDH (CST, #5174, 1:1000) and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, anti-rabbit [111–035-047], 1:5000; anti-mouse [315–035-047], 1:5000).
4
Lung Histopathology and Mucin Expression
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The upper lobe of left lung without lavage was immediately fixed with 10% formaldehyde for 48 h, embedded in paraffin, sliced into 4 μm thick sections, and stained with hematoxylin and eosin solution (H&E) or Alcian blue periodic acid Schiff (AB/PAS) for histopathologic examination and goblet cell metaplasia of bronchial epithelium.
For analyzing the effects of GFDHP on mucus secretion, the Mucin5ac (Muc5ac) protein expression levels in the airway epithelium were performed by immunohistochemistry (IHC) analysis [18 (link)]. Change in the MAPK pathway in the airway was investigated on the basis of detecting Muc5ac regulatory proteins including extracellular signal-regulated kinase (ERK)/p-ERK/specificity protein1(SP1) through IHC. After antigen was repaired and endogenous peroxidase was inactivated and blocked, the sections were incubated overnight at 4°C with anti-Muc5ac (Abcam, USA, dilution 1 : 200), anti-ERK (dilution 1 : 200), anti-p-ERK (dilution 1 : 500), and anti-SP1 (dilution 1 : 200) antibodies (Proteintech, China) successively. The sections were then incubated with horseradish peroxidase- (HRP-) conjugated second antibodies and developed with 3, 3-diaminobenzidine tetrahydrochloride. The degree of brown stains reflected the expression levels of the target proteins.
For analyzing the effects of GFDHP on mucus secretion, the Mucin5ac (Muc5ac) protein expression levels in the airway epithelium were performed by immunohistochemistry (IHC) analysis [18 (link)]. Change in the MAPK pathway in the airway was investigated on the basis of detecting Muc5ac regulatory proteins including extracellular signal-regulated kinase (ERK)/p-ERK/specificity protein1(SP1) through IHC. After antigen was repaired and endogenous peroxidase was inactivated and blocked, the sections were incubated overnight at 4°C with anti-Muc5ac (Abcam, USA, dilution 1 : 200), anti-ERK (dilution 1 : 200), anti-p-ERK (dilution 1 : 500), and anti-SP1 (dilution 1 : 200) antibodies (Proteintech, China) successively. The sections were then incubated with horseradish peroxidase- (HRP-) conjugated second antibodies and developed with 3, 3-diaminobenzidine tetrahydrochloride. The degree of brown stains reflected the expression levels of the target proteins.
5
Western Blot Analysis of GC Cell Proteins
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In a radio-immunoprecipitation assay (RIPA) lysis buffer, the proteins were extracted from GC cells. Ten percents of polyacrylamide sodium dodecyl sulfate (SDS) gels (SDS-PAGE) were used to separate equivalent amounts of proteins. The proteins were then blotted onto membranes of polyvinylidene fluoride (Amersham, UK). After membranes were blocked at RT (37°C) in bovine serum albumin (5%) for 1 h, primary antibodies were used to culture the membranes overnight. After being cultured, they were placed in secondary antibodies conjugated with human resource planning (Perbio Science, Belgium), and incubated at RT (37°C) for 1 h. The system of enhanced chemiluminescence immunodetection (Immobilon, USA) was used to visualize immunoreactive proteins. The antibodies for Western blotting were as follows: anti-KIF22 (13403-1-AP; Proteintech Group, USA), anti-Phospho-MEK1/2 (CST: #9154; Proteintech Group), anti-MEK (CST: #8727; Cell Signaling Technology, USA), anti-Phospho-ERK1/2 (CST: #4370; Proteintech Group), anti-ERK (16443-1-AP; Proteintech Group), anti-Cyclin A2 (18202-1-AP; Proteintech Group), anti-Cyclin B1 (55004-1-AP; Proteintech Group), anti-Cyclin D1 (26939-1-AP; Proteintech Group), anti-P21 (10355-1-AP; Proteintech Group), and β-actin (20536-1-AP; Proteintech Group).
6
Protein Expression Analysis of Pulmonary Artery Cells
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The PAs homogenate tissue and collected HPASMCs and HPAECs were dissolved in proteolytic buffer for 30 minutes. After 30 minutes, the lysed PAs homogenate tissue and cell proteins were centrifuged at 13 500 g for 15 minutes, and then the protein concentration was measured using a BCA kit. Thirty micrograms of cell lysate from each sample were used for SDS‐PAGE (Bio‐Rad Laboratories), and Western blotting were analysed according to the protocol as described previously.23 The chosen antibodies included anti‐NDUFA4L2 (Catalogue number: 16480‐1‐AP; ProteinTech), anti‐HIF1α (Catalogue number: AF1009; Affinity), anti‐PCNA (Catalogue number: 10205‐2‐AP; ProteinTech), anti‐cyclin A (Catalogue number: 13295‐1‐AP; ProteinTech), anti‐cyclin E (Catalogue number: 11554‐1‐AP; ProteinTech), anti‐ERK (Catalogue number: 16443‐1‐AP; ProteinTech), anti‐p‐ERK (Catalogue number: AF1015; Affinity), anti‐5‐LO (Catalogue number: AF4699; Affinity), anti‐p‐5‐LO (Catalogue number: AF8359; Affinity), anti‐p38 (Catalogue number: 14064‐1‐AP; ProteinTech), anti‐p‐p38 (Catalogue number: 4511T; Cell Signaling), anti‐JNK (Catalogue number: 51151‐1‐AP; ProteinTech) and anti‐p‐JNK (Catalogue number: 4668S; Cell Signaling) et al. After extensive washes membranes with TBS‐T, the ECL luminescent solution is added for exposure and development.
7
Antibody Detection Protocol for Cellular Signaling
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Anti-CD40 (#28158-1-AP, 1 : 1000), anti-AKT (#10176-2-AP, 1 : 1000), anti-GAPDH (#10494-1-AP, 1 : 10000), and anti-ERK (#16443-1-AP, 1 : 1000) antibodies were purchased from Proteintech (Hubei, Wuhan, China). anti-AKT-phosphor-Ser473 (#4060, 1 : 1000) and NF-κB p65 (#8242, 1 : 1000) antibodies were obtained from Cell Signaling Technology (Massachusetts, USA). Anti-phosphor-ERK (#461212082, 1 : 1000) was bought from Sigma (St. Louis, MO, USA). Anti-phosphor-STAT3-Tyr705 (#abs130918, 1 : 1000) was bought from absin (China). And anti-STAT3 (#GB11176, 1 : 1000) and anti-ICAM-1 (#GB11106, 1 : 1000) were obtained from Servicebio (Wuhan, China).
8
Evaluating Ras Signaling Modulation
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METH was provided by Ningbo Public Security Bureau. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich. All drugs were dissolved in saline (0.9% NaCl) and administered to cells directly or via intraperitoneal injection. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DMSO were purchased from MedChemExpress. The BrdU cell proliferation kit was purchased from BioVision. The primary and secondary antibodies for Western blotting, including anti-Ras, anti-β-actin, anti-ERK, anti-pERK, anti-MEK, and anti-pMEK, were purchased from Proteintech. Polyvinylidene fluoride, blocking buffer, and ECL were purchased from Millipore. The Active Ras Pull-Down and Detection Kit was purchased from Thermo Fisher Scientific.
9
Western Blot Analysis of Inflammasome
10
Western Blotting with Diverse Antibodies
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Western blotting was carried out as described previously 15 (link), 21 (link), 22 (link). Anti-Bcl-2, anti-Mcl-1, anti-PARP1, anti-cytochrome C, anti-ERK, anti-p-ERK, anti-Bim, anti-TSHR, anti-HK2, anti-Bax, anti-cyclin D1, and anti-GAPDH antibodies were purchased from ProteinTech (Chicago, IL, USA); anti-GLUT1 (NB110-39113SS) antibody was acquired from Novusbio. Anti-Beclin1 (#3738), anti-p62 (#8025), anti-p27 Kip1 (#3688), anti-Bcl-xl (#2764), anti-LC3A/B (#12741), anti-Caspase-3 (#9662), anti- Caspase-9 (#9502), anti-Caspase-8 (#9746) antibodies were purchased from Cell Signaling Technology. For immunoprecipitation, cells were treated for 24 hours with Vemurafenib before harvesting. NP40 lysates and BRAF immunoprecipitates were prepared according to the previous publication using rabbit anti-BRAF antibody (Santa Cruz Biotechnology, sc-5284) and analyzed by immunoblot using mouse anti-CRAF antibody (Santa Cruz Biotechnology, sc-227) or inversely 23 (link).
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