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).
Ab7952
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab7952 is a laboratory equipment product manufactured by Abcam. It is a tool used for scientific research purposes. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab5694, by Abcam (2 mentions)
Ab199380, by Abcam (2 mentions)
Ab180163, by Abcam (2 mentions)
Ab4822, by Abcam (2 mentions)
Bx ucb fluorescent microscope, by Olympus (2 mentions)
Nl006, by R&D Systems (2 mentions)
Dapi containing mounting media, by Vector Laboratories (2 mentions)
Ab14106, by Abcam (1 mentions)
Ab50011, by Abcam (1 mentions)
Ab52866, by Abcam (1 mentions)
Ab47363, by Abcam (1 mentions)
Ab189403, by Abcam (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab46794, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Abt128, by Merck Group (1 mentions)
Ab32072, by Abcam (1 mentions)
Ab4821, by Abcam (1 mentions)
Ab178867, by Abcam (1 mentions)
Ab47943, by Abcam (1 mentions)
9 protocols using ab7952
1
Protein Expression Analysis in Cells
2
Protein Expression Analysis in Cells
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Protein samples of the cells were extracted by lysing the cells with radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The protein samples were quantified using the BCA Protein Assay Kit (Beyotime) and the same amount of samples were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands on the gel were transferred to a polyvinylidene fluoride membrane and blocked in 5% skim milk for 2 h at room temperature and then the blot was incubated in the specific primary antibodies for TSLP (ab47943), skeletal muscle actin (α-SMA, ab5694), collagen I (ab6308), MAPK7 (ab40809), extracellular signal-regulated kinase 1 (ERK1, ab180163), phospho-ERK1 (p-ERK1, ab24157), p38 (ab7952), p-p38 (ab178867), c-Jun N-terminal kinase 1 (JNK1, ab199380), and p-JNK1 (ab47337), purchased from Abcam (Cambridge, UK), overnight at 4°C. β-actin (ab189073) was used as an endogenous reference. After washing, the blot was incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Positive signals were developed using the ECL Plus Western Blotting Substrate (Thermo Scientific). Band densities for each sample were analyzed using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD).
3
Western Blot Analysis of Signaling Proteins
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Cells were lysed on ice using a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins were collected, electrophoresed via 10% SDS-PAGE, then transferred to polyvinylidene fluoride membranes (Roche Diagnostics GmbH, Mannheim, Germany). Then, the membranes were incubated at 4°C overnight with the following primary antibodies: Anti-p38 at a 1:1,000 dilution (ab7952; Abcam, Cambridge, UK), anti-phosphorylated (p)-p38 at a 1:500 dilution (ab4822; Abcam), anti-TGF-β1 at a 1:500 dilution (ab92486; Abcam), anti-fibronectin at a 1:500 dilution (ab2413; Abcam) and β-actin at a 1:1,000 dilution (sc-130656; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The species-specific horseradish peroxidase-conjugated secondary antibodies (ZDR-5306; ZSGB-Bio, Beijing, China) were added at a 1:500 dilution and incubated for 1 h at room temperature to the membranes, and immunoreactive protein bands were detected using a Western Bright Enhanced Chemiluminescence detection system (Advansta, Inc., Menlo Park, CA, USA).
4
Protein Expression Analysis by Western Blot
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Equal amounts of protein samples were extracted from cells and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% fat-free milk, then incubated with antibodies against ACE2 (1:3,000, ab108252, Abcam, US), VE-cadherin (1:1,000, ab33168, Abcam), EphA2 (1:1,000, 6997s, CST, USA), AKT (1:1,000, 4691, CST), p-AKT (1:2,000, 4060, CST), p38 (1:2,000, ab7952, Abcam), p-p38 (1:1,000, 4511, CST), Nodal (1:2,000, ab55676, Abcam), Notch4 (1:2,000, ab184742, Abcam), and Actin (1:2,000, ab8226, Abcam) overnight at 4°C, and the corresponding secondary antibodies (1:5,000, Thermo Fisher, USA) for 1 h at room temperature (RT). The blots were examined by an enhanced chemiluminescence detection kit (Millipore). Actin was used as a loading control, and all experiments were repeated three times independently. Expression quantification of proteins was performed using ImageJ software (National Institutes of Health, USA).
5
Immunohistochemistry of p38 in Mouse Retina
6
Regulation of PDCD4 via miR-16 and MAPK
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RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and ox-LDL were purchased from Invitrogen (Shanghai, China). miR-16 mimic, miR-16 inhibitor and siRNA targeting PDCD4 (PDCD4 siRNA) were purchased from GenePharma (Shanghai, China), as well as their negative control (NC) oligonucleotide duplex that did not target any gene. Antibodies against p38 (ab7952), phosphorylated (p-)p38 (ab4822), extracellular signal-regulated kinase (ERK; ab180163), p-ERK (ab131438), c-Jun NH2-terminal kinase (JNK; ab199380), p-JNK (ab18680), p65 (ab90532) and PDCD4 (ab79405) were obtained from Abcam (Cambridge, MA, USA).
7
Analyzing Ptch1 Knockout MEFS Expression
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Ptch1−/− MEFS containing the integrated hPtch1 variants were trypsinized and analyzed compared to an uninfected (control) population using fluorescence activated cell sorting. A population of cells with fluorescence from the integrated hPtch1-mVenus-1D4 construct was clearly visible. This population was used to set a sorting gate for the cells containing the hPtch1 gene. Approximately 250,000 cells were sorted and then plated into a 6-cm dish. Once these cells were grown to a sufficient density, they were seeded into additional plates for Western blot analysis (41 (link)) to confirm expression of the hPTCH1-mVenus-1D4 gene. An anti–green fluorescent protein antibody (Novus Biologicals, catalog no. NB600-308, RRID: AB_10003058) was used to detect PTCH1 expression levels, and anti-P38 (Abcam, catalog no. ab7952, RRID: AB_306166 ) was used as a gel loading control.
8
Immunohistochemistry of p38 in Mouse Retina
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Mouse retina cryosections (10 μm) were fixed in 4% paraformaldehyde and blocked with 10% normal goat serum. The sections were rinsed with PBS, permeabilized with 0.1% Triton X-100, and incubated over night with anti-p38 antibody (rabbit polyclonal, ab7952, Abcam; Cambridge, MA). The slides were rinsed with PBS, and incubated with the anti-rabbit-FITC conjugated secondary antibody (NL006; R&D systems; Minneapolis, MN) for 1 hour [26 (link)]. After rinsing the slides with PBS, the sections were mounted with DAPI-containing mounting media (Vector Laboratories; Burlingame, CA), and imaged with an Olympus BX-UCB fluorescent microscope (20X magnification). The fluorescence intensity was quantified by using ImageJ software (version 1.44; NIH). Cryosections processed under similar conditions, except being incubated with the primary antibody, served as controls.
9
Protein Expression and Analysis
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Protein extraction and western blot analysis were conducted as described previously (20 (link)). The primary antibodies used were anti-MAPK1 (Abcam, Cambridge, UK, ab102930, 1:500), anti-p38 MAPK (Abcam, ab7952, 1:800), anti-B-cell lymphoma (Bcl)-2 (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany, SAB4300339, 1:500), anti-Raf-1 (Abcam, ab32025, 1:800) and anti-β-actin (Santa Cruz Biotechnology, Inc., sc-8432, 1:500).
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