Western blotting was performed from neonatal rat cardiomyocytes using standard procedures. Antibodies used were METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling Technology) and GAPDH (Fitzgerald Industries). Masson’s trichrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining was performed using α-Actinin antibodies (Sigma Aldrich) and cell area was quantified using CellProfiler following published methods.24 (link) Detection of the cell membrane was performed using FITC-conjugated Wheat Germ Agglutinin (Sigma Aldrich). Cross-sectional area was measured using ImageJ. RNA was extracted from neonatal rat cardiomyocytes or mouse hearts using Trizol and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Selected genes and m6A peaks were analyzed by real-time PCR using SYBR green (Applied Biosystems). Quantified mRNA expression was normalized to Rpl7 (Ribosomal Protein L7), and expressed relative to controls.
Mapk14
Manufactured by Cell Signaling Technology
MAPK14 is a mitogen-activated protein kinase that plays a key role in the cellular response to various environmental stressors. It is involved in the regulation of cell growth, differentiation, and apoptosis. MAPK14 is a widely studied enzyme in cell signaling research.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Mettl3, by Fortis Life Sciences (2 mentions)
Map3k6, by Novus Biologicals (2 mentions)
α actinin antibody, by Merck Group (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Map4k5, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Biosynth (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Fitc conjugated wheat germ agglutinin, by Merck Group (1 mentions)
Fluorescein isothiocyanate conjugated wheat germ agglutinin, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mapkapk3, by Cell Signaling Technology (1 mentions)
Gapdh sc 47724, by Santa Cruz Biotechnology (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Thr180 tyr182 p p38mapk, by Cell Signaling Technology (1 mentions)
Amersham imager 600 digital imagingsystem, by GE Healthcare (1 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
6 protocols using mapk14
1
Western Blotting and Immunostaining of Neonatal Rat Cardiomyocytes
2
Cardiac Protein and Gene Expression Analysis
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Western blotting was performed from neonatal rat cardiomyocytes using standard procedures. Antibodies used were METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling Technology), and GAPDH (Fitzgerald Industries). Masson trichrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining was performed using α-actinin antibodies (Sigma-Aldrich), and cell area was quantified using CellProfiler following published methods.24 (link) Detection of the cell membrane was performed using fluorescein isothiocyanate–conjugated wheat germ agglutinin (Sigma-Aldrich). The cross-sectional area was measured using ImageJ. RNA was extracted from neonatal rat cardiomyocytes or mouse hearts using Trizol, and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Selected genes and m6A peaks were analyzed by real-time polymerase chain reaction using SYBR green (Applied Biosystems). Quantified mRNA expression was normalized to Rpl7 (ribosomal protein L7) and expressed relative to controls.
3
Protein Quantification and Western Blot Analysis
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Ten micrograms of protein from each sample
was mixed with 5× Laemmli buffer with 5% β-mercaptoethanol,
heated for 5 min at 100 °C, and separated on 10% sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis gels. Following electrophoresis,
the proteins were transferred to Immobilon-P transfer membranes (Sigma-Aldrich)
using the Trans-Blot TurboTM Transfer System (Bio-Rad Laboratories).
Membranes were visualized with Ponceau Red (FlukaTM Analytical). Membranes
were probed with the following antibodies purchased from Cell Signaling
Technology: MAPKAPK3 (#7421), MAPK14 (#9218), Thr180/Tyr182-P-p38
MAPK (#4511), and anti-rabbit IgG-HRP (#7074). GAPDH (sc-47,724) was
purchased from Santa Cruz. The Amersham Imager 600 digital imaging
system (GE Healthcare) was used for image acquisition and Quantity
One v4.6.3 (Bio-Rad Laboratories) for band densitometry. All western
blots are presented as cropped images, with full scans of blots provided
inFigure S4 .
was mixed with 5× Laemmli buffer with 5% β-mercaptoethanol,
heated for 5 min at 100 °C, and separated on 10% sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis gels. Following electrophoresis,
the proteins were transferred to Immobilon-P transfer membranes (Sigma-Aldrich)
using the Trans-Blot TurboTM Transfer System (Bio-Rad Laboratories).
Membranes were visualized with Ponceau Red (FlukaTM Analytical). Membranes
were probed with the following antibodies purchased from Cell Signaling
Technology: MAPKAPK3 (#7421), MAPK14 (#9218), Thr180/Tyr182-P-p38
MAPK (#4511), and anti-rabbit IgG-HRP (#7074). GAPDH (sc-47,724) was
purchased from Santa Cruz. The Amersham Imager 600 digital imaging
system (GE Healthcare) was used for image acquisition and Quantity
One v4.6.3 (Bio-Rad Laboratories) for band densitometry. All western
blots are presented as cropped images, with full scans of blots provided
in
4
Western Blot Analysis of Osteoclast Markers
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CD14+PBMCs were lysed on ice using RIPA (Sigma-Aldrich; Merck KGaA) containing PMSF and protease inhibitors (Roche Diagnostics GmbH). Protein concentration was determined by a BCA kit (Thermo Fisher Scientific, Inc.). Proteins (20 µg per lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). Membranes were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 1 h and were incubated with primary antibodies against GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), MAPK14 (1:1,000; cat. no. 8690; Cell Signaling Technology, Inc.), TRAP (1:1,000; cat. no. ab65854; Abcam), NFATC1 (1:1,000; cat. no. ab25916; Abcam), CTSK (1:1,000; cat. no. ab37259; Abcam), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.) and p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.) at 4˚C overnight. Membranes were then incubated with anti-rabbit (1:2,000; cat. no. 7074) or anti-mouse HRP-conjugated secondary antibody (1:2,000; cat. no. 7076; both from Cell Signaling Technology, Inc.) at room temperature for 1 h. Enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.) was used to detect the signal on the membrane. Densitometry analysis was performed using Image J software (v1.50; National Institutes of Health).
5
Western Blot Protein Analysis
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Cell lysates were prepared using 1 × RIPA lysis buffer (Biosaesang) supplemented with a protease inhibitor (Roche), cleared by centrifugation at 12,000g, separated by SDS–PAGE, and transferred onto PVDF membranes (Millipore). Primary antibodies were incubated as indicated; overnight at 4 °C in 1 × TBST with 5% skim milk or 5% BSA; PTBP1 (1:1,000, Invitrogen), MAPK14 (1:1,000, Cell Signaling Technology), PCSK9 (1:1,000, Cayman Chemical Company), γ-tubulin (1:1,000, Cell Signaling Technology) and α-tubulin (1:1,000, Cell Signaling Technology).
6
EGFR Signaling Pathway Inhibition Assay
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Inhibitors afatinib, gefitinib, erlotinib, selumetinib, sapanisertib, and rapamycin were purchased from Selleckchem. EGTA and lanthanum(III) Chloride hydrate were purchased from Sigma-Aldrich. Cell lines NCI-H1975, NCI-H1650, and HCC827 were derived from the ATCC, and PC9 was derived from Sigma. EGFR-pTyr1068, Paxillin-pTyr31, and Actin antibodies were from Abcam. EGFR, Myosin-IIa, Myosin-IIa-pSer1943, Myosin-IIc, Erk1/2, Erk1/2-pThr208/pTyr206, RPS6, RPS6-p-Ser235/Ser236, MAPK14, Paxillin, Paxillin-pTyr118, FAK, FAK-pTyr576/577, and FAK-pTyr397 antibodies were from Cell Signaling Technology. GAPDH was derived from GeneTex.
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