The content of PC1, SM22, ACTA2, CNN1, p38, ERK, JNK, PI3K and Myc, CyclinD1, and the phosphorylation of p38, ERK, JNK and PI3K were determined by Western blot. Preparation of whole cell lysates and tissue homogenates, and the immunoblotting assay were performed according to previously described procedures.18 (link) The primary antibodies were obtained from the following sources: anti-Polycystin-1 (ABT128; Millipore); anti-SM22 alpha (ab14106; Abcam, Cambridge, UK); anti-alpha smooth muscle Actin (ab5694; Abcam); anti-Calponin (ab46794; Abcam); anti-alpha Tubulin (ab52866; Abcam); anti-p38 (phospho Y182) (ab47363; Abcam); Anti-p38 (ab7952; Abcam); Anti-ERK1 (pT202/pY204) + ERK2 (pT185/pY187) (ab50011; Abcam); Anti-ERK1 + ERK2(ab17942; Abcam); Anti-JNK1+JNK2 (phospho T183 + Y185) antibody (ab4821); Anti-JNK1+JNK2 (ab37228; Abcam); Anti-PI3K p85 (phospho Y607); Anti-PI3K p85 (ab189403; Abcam); anti-myc (ab32072; Abcam) and anti-Cyclin D1 (ab134175; Abcam).
Ab47363
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab47363 is a primary antibody designed for use in various immunoassays. The product details and specifications are available on the Abcam website.
Automatically generated - may contain errors
Lab products found in correlation
Ab31828, by Abcam (9 mentions)
Ab17942, by Abcam (5 mentions)
Ab124956, by Abcam (4 mentions)
Ab170099, by Abcam (4 mentions)
Ab16502, by Abcam (4 mentions)
Ab50011, by Abcam (3 mentions)
Ab86299, by Abcam (3 mentions)
Ab208035, by Abcam (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab179461, by Abcam (2 mentions)
Ab214362, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Abt128, by Merck Group (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab14106, by Abcam (1 mentions)
Ab52866, by Abcam (1 mentions)
Ab7952, by Abcam (1 mentions)
Ab189403, by Abcam (1 mentions)
18 protocols using ab47363
1
Protein Expression Analysis in Cells
2
Immunohistochemical Analysis of MAPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sections of colon tissue were deparaffinized by xylene, hydrated with gradient ethanol, incubated with 0.1% Tritonx-100 for 30 min, and rinsed 3 times with PBS for 5 min each. The sections were blocked by 5% BSA and 10% sheep serum successively for 30 min. The sections were incubated separately with primary antibody ERK1/2 (ab17942, Abcam), p-ERK (ab192591, Abcam), p38 (ab31828, Abcam), p-p38 (ab47363, Abcam), JNK (ab208035, Abcam), and p-JNK (ab124956, Abcam) in a wet box at 4°C overnight, washed 3 times with PBS for 5 min per time and second antibody at 37°C for 40 minutes, and washed with PBS 3 times for 5 minutes per time. Horseradish peroxidase-labeled streptavidin protein was added dropwise at 37°C for 40 minutes. The sections were rinsed 3 times with PBS for 5 minutes each time, developed by DAB, microscopically observed, and photographed.
3
Western Blot Assay for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein used for the western blot assay was extracted by using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China), and the concentrations of these protein samples were quantified by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Appleton, WI, USA). The equivalent proteins were separated on gels by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% BSA, these membranes were incubated with primary antibodies of p53 (ab131442), CyclinD1 (ab16663), Bax (ab182733), Bcl-2 (ab32124), pro-caspase-3 (ab32150), cleaved-caspase-3 (ab2302), MyD88 (ab133739), total (t)-IκBα (ab178846), p-IκBα (ab133462), t-p65 (ab32536), p-p65 (ab86299), t-p38MAPK (ab170099), p-p38MAPK (ab47363), and β-actin (ab6276) (from Abcam, Cambridge, UK) at 4°C overnight. Subsequently, membranes were washed with TBS with Tween 20 (TBST), and the secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; ab205718, 1:2,000, Abcam) was added to incubate for 1 h at room temperature. After this, 200 μL of Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was supplemented to cover the surface of the membrane, and the signals were captured. The intensity of the bands was quantified using Image Lab software (Bio-Rad).
4
YfeA modulates signaling pathways in RAW 264.7 macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 macrophages were treated with or without YfeA (20 µg/mL) for 12 h, the total cellular protein was extracted using a Protein Extracting Kit (Solarbio, Beijing, China). Aliquots corresponding to 50 µg of each sample were analyzed using Western blotting (WB). To analyze the YfeA-induced signal transduction residue phosphorylation, the primary monoclonal antibodies, including Erk1/2 (rabbit, 42/44 kDa; 1:2000; Abcam ab184699), p-Erk1/2 (rabbit, p-ERK1: Thr202/Tyr204, p-ERK2: Thr185/Tyr187; 42/44 kDa; 1:2000; Abcam ab278538), p38-MAPK (rabbit, 41 kDa; 1:1000; Abcam ab31828), p-p38-MAPK(rabbit, Tyr182; 41 kDa; 1:500; Abcam ab47363), JNK (rabbit, 48 kDa; 1:1000; Abcam ab179461), p-JNK (rabbit, Tyr185/223; 48 kDa; 1:10,000 Abcam ab76572), p65 (NF-κB; rabbit, 60 kDa; 1:2000; Abcam ab16502), p-p65 (NF-κB; rabbit, Ser536; 60 kDa; 1:5000; Abcam ab86299), TLR2 (rabbit, 89 kDa; 1:500; Abcam ab213676), and TLR4 (rabbit, 89 kDa; 1:500; Abcam ab13556) were applied at this stage.
5
Detailed Protocol for Inflammatory Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetonitrile (34998) and formic acid (00940) for LC-MS were obtained from Merck (WGK, Germany). InComplete Freund’s adjuvant (IFA), Complete Freund’s adjuvant (CFA), and bovine type II collagen were obtained from Chondrex, Inc. (Redmond, Wash.). ELISA kits for detections of IL-1β (10 × 96t, 88-7013-88), IL-6 (2 × 96t, 88-7064-86), TNF-α (2 × 96t, 88-7324-22), IL-4 (2 × 96t, 88-7044-22) and IL-10 (2 × 96t, 88-7105-22) were purchased from Invitrogen (Karlsruhe, Germany). Anti-IKKα (ab32041, 1:1000), Anti-IKKβ (ab124957, 1/1000), anti-IKKα/β (ab194528, 1:1000), anti-NF-κB p65 antibody (ab16502, 1:1000), anti-P-NF-κB p65 (phospho S536) antibody (ab76302, 1:1000), anti-P38 (ab122517, 1:1000) antibody, anti-P-P38 (phospho Y182) antibody (ab47363, 1:1000), anti-Histone H3 antibody (ab1791, 1:1000) and secondary antibodies were obtained from Abcam (Cambridge, United Kingdom). Anti-IκBα, anti-P-IκBα (phospho S36/32), anti-P-ERK (Thr202/Tyr204), Anti-ERK, anti-JNK, anti- P- JNK (Thr183/Tyr185), anti-STAT6 and anti-P-STAT6 antibodies were obtained from cell signaling technology (Boston, United States). All other reagents and chemicals used were of standard biochemical quality.
6
Membrane Protein Extraction and MAPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The membrane protein extractions from the aortic media (VSMCs were the only cell type in this layer) or cultured VSMCs were carried out by the Plasma Membrane Protein Isolation Kit (Abcam, UK) according to the manufacturer's instructions. Total proteins were also extracted, and western blot was performed as described in our previous reports [12 (link), 13 (link)]. The expressions of NOX subunits and mitogen-activated protein kinase (MAPK) signaling pathway were detected using their specific antibodies: anti-NOX1 (1 : 2000, ab55831, Abcam, Cambridge, UK), anti-NOX2 (1 : 500, ab80508, Abcam, Cambridge, UK), anti-p47phox (1 : 1000, ab795, Abcam, Cambridge, UK), anti-NADPH oxidase organizer 1 (anti-NOXO1, 1 : 500, sc-390927, Santa Cruz Biotechnology Co., Ltd., Japan), anti-Rac1 (1 : 1000, sc-95, Santa Cruz Biotechnology Co.), anti-p38 (1 : 1000, ab31828, Abcam), anti-phospho-p38 (p-p38, 1 : 1000, ab47363, Abcam), anti-ERK1/2 (1 : 1000, ab17942, Abcam), anti-phospho-ERK1/2 (p-ERK1/2, 1 : 1000, 9101, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-JNK (1 : 1000, ab179461, Abcam), and anti-phospho-JNK (p-JNK, 1 : 1000, ab124956, Abcam). To ensure equal protein loading, β-actin (1 : 5000, ab8226; Abcam) and Na+/K+-ATPase (1 : 100000, ab76020, Abcam) were used as loading controls for the cytoplasm and plasma membrane, respectively.
7
Western Blot Analysis of Apoptosis Markers in Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from the primary cardiomyocytes in RIPA buffer (1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.0; Solarbio, China) supplemented with a protease inhibitor cocktail (Cat: I3786-1ML, Sigma). The cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a PVDF membrane (Millipore Corporation, USA). After blocking with 8% milk in PBS, pH 7.5, the membranes were incubated with the following specific primary antibodies of Bax (ab32503), Bcl-2 (ab59348), cytochrome C (ab13575), Smac/Diablo (ab32023), cleaved-capase-3 (ab13847), cleaved-capase-9 (ab2324), p-p38 (ab47363), p38 (ab31828), p-ERK (ab214362), and ERK1/2 (ab196883; all at a dilution of 1:1000, Abcam, UK). After overnight incubation, the appropriate HRP-conjugated anti-rabbit IgG secondary antibody (ab205781, Abcam, all at a dilution of 1:5000) was subsequently applied and immunodetection was achieved using the ECL Plus detection system (Millipore Corporation) according to the manufacturer's instructions. Band intensity was quantified using Image Lab™ Software (Bio-Rad, China). GAPDH (ab8245, Abcam) was used as an internal control.
8
Western Blot for Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The detailed process of Western blot was described according to our previous study (18 (link)). The primary antibodies included NF-κB (bs-0465R, 1:1000, Bioss), p-NFκB (#3033, 1:1000, CST), p38 (ab170099, 1:1000, abcam), p-p38 (ab47363, 1:500, abcam), JNK (#9252, 1:1000, CST), p-JNK (#4668, 1:1000, CST), and β-actin (66009-1-Ig, 1:5000, Proteintech).
9
Western Blot Analysis of SEMA4C and MAPK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysis buffer was used to extract proteins from HCC tissues or cells. After centrifugation at 12,000 × g, 4°C for 30 min, the supernatant of tissues or cells was measured by BCA kit. Protein specimens (50 µg) were added onto SDS-PAGE and electrophoresed at 60 V. Proteins were then transferred to nitrocellulose filter membranes (NC). Subsequently, the membranes were blocked with skim milk (5-10%) at 37°C for 1 h and then incubated with the primary antibodies against SEMA4C (1:500; sc-136445; Santa Cruz Biotechnology, Inc.), p38 (1:1000, ab170099; Abcam), p-p38 (1:1000, ab47363; Abcam), MAPK (1:1000, ab185145; Abcam), p-MAPK (1:2000, 4370; Cell Signaling Technology, Inc.,) at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 h at 37°C. GAPDH (1:2000; ab181602; Abcam) was used as the loading control. Finally, enhanced chemiluminescence kit (ECL; EMD Millipore) was used to detect the signals.
10
Protein Expression Analysis in AF Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After AF cells were cultured in the conditioned medium, they were washed with sterile PBS for two times. Total protein samples were extracted by RIPA solution (Beyotime, China), and then quantified using a BCA Protein Assay Kit (Beyotime, China). After protein samples were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), they were transferred on to the polyvinylidene fluoride (PVDF) membranes (Beyotime, China). Then, the PVDF membranes were sequentially immunostained with primary antibodies (GAPDH: Abcam, ab181602; cleaved caspae-3: Cell Signaling Technology, #9664; Cleaved PARP: Cell Signaling Technology, #94885; JNK: Cell Signaling Technology, #9255; p-JNK: Cell Signaling Technology, #9252; p38 MAPK: Abcam, ab31828; p-p38 MAPK: Abcam, ab47363) overnight at 4°C and the secondary antibodies for 2 h at room temperature. After the protein bands were detected by enhanced ECL reagent (BeyoECL Plus, Betyotime, China), the immunoreactive protein bands were quantified by densitometry (Image J software, Wayne Rasband, National Institutes of Health, USA) with a loading control of GAPDH.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!