Total protein was collected from gastric cancer cell lines and xenograft from nude mice using RIPA lysis buffer for 30 min at 4°C containing protease inhibitors, and the homogenates were centrifuged at 12,000 × g for 20 min at 4°C. Protein concentration was estimated by a BCA Protein kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of proteins (25 μg) were separated by 10–15% SDS-PAGE and transferred into nitrocellulose membrane (Millipore). After blocking with 5% fat-free milk overnight at 4°C, the blots were incubated with anti-PRAKK1 (Abcam), anti-PCNA (Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p-ERK1 (Abcam), anti-ERK1 (Abcam), anti-p-STAT3 (Abcam), anti-STAT3 (Cell Signaling Technology), anti-p-JNK1 (Abcam), anti-JNK1 (Abcam), anti-p-Akt (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology) antibody overnight at 4°C. The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000; Beyotime) for 1 h at 37°C. The membranes were developed using an enhanced chemiluminescence (ECL) kit (Applygen Technologies, Beijing, P.R. China) following the manufacturer’s instructions.
Anti jnk1
Manufactured by Abcam
Sourced in United States
Anti-JNK1 is a primary antibody that recognizes the c-Jun N-terminal kinase 1 (JNK1) protein. JNK1 is a member of the mitogen-activated protein kinase (MAPK) family and plays a role in cellular processes such as proliferation, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Anti oct4, by Abcam (3 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti sox2, by Abcam (2 mentions)
Anti afp, by Affinity Biosciences (2 mentions)
Anti heppar1, by Novus Biologicals (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Bca protein kit, by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti p stat3, by Abcam (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Beyotime (1 mentions)
Ecl kit, by Applygen (1 mentions)
Anti nanog, by Abcam (1 mentions)
Anti survivin, by Abcam (1 mentions)
Anti ampk, by Abcam (1 mentions)
5 protocols using anti jnk1
1
Gastric Cancer Protein Expression Analysis
2
Protein Expression Analysis in fMSCs, hGCs, and Ovarian Tissue
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fMSCs, hGCs, and ovarian tissues were harvested for protein extraction. Western blotting was performed as previously described [12 (link)]. The primary antibodies used for fMSCs were anti-Oct4, anti-Nanog, anti-Rex1, and anti-β-Actin, all purchased from Abcam (USA). The primary antibodies used for hGCs and the ovarian tissues were anti-SURVIVIN, anti-BCL2, anti-CASPASE-3, anti-CASPASE-9, anti-MT1, anti-JNK1, anti-PCNA, anti-AMPK, anti-β-Actin, and anti-GAPDH, all purchased from Abcam (USA).
3
Western Blot Analysis for Protein Expression
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Proteins were extracted with a RIPA buffer containing phenylmethane sulfonyl fluoride (Sigma, St. Louis, MO, USA) and a protease inhibitor cocktail (Sigma). Then, 20 μg of protein was separated by 8–12% Tris-acrylamide gels and incubated with primary antibodies overnight at 4°C. Afterwards, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The primary antibodies used were as follows: anti-AFP (1:500; Affinity), anti-GAPDH (1:1,000; Affinity, Cincinnati, OH, USA), anti-HEPPAR1 (1:500; Novus Biologicals Centennial, CO, USA), anti-JNK1 (1:1,000; Abcam, Cambridge, UK), anti-OCT4 (1:1,000; Abcam), and anti-SOX2 (1:1,000; Abcam). The results were detected through an ECL reagent (Millipore, Billerica, MA, USA) and captured by an electrophoresis gel imaging system (ChemiScope 6000, CLIX, Shanghai, China). The experiments above were performed in triplicate.
4
Proteomic Analysis of Photodynamic Therapy Effects
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Cancer cells treated with or without photosensitizers and irradiation were washed with ice‐cold PBS for 3 times to remove the residual medium. Then cells were lysed in lysis buffer to harvest total proteins, and then detected by Nanodrop to measure the protein concentrations. The protein samples were separated on 6%, 10% and 12% (v/v) SDS polyacrylamide gels and transferred into polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin or dehydrated milk, the membranes were incubated with primary antibodies as following, anti‐Bax (abcam), anti‐Bcl‐2 (abcam), anti‐Caspase‐3 (abcam), anti‐Cyto C [EPR1327] (abcam), anti‐PI3K p85α [EPR18702] (abcam), anti‐PI3K p110β [EPR5515(2)] (abcam), anti‐NF‐κB [E379] (abcam), anti‐IκB [E130] (abcam), anti‐JNK1 + JNK2 + JNK3 [EPR16797‐211] (abcam), anti‐JNK1 + JNK2 + JNK3 (phospho T183 + T183 + T221) [EPR5693] (abcam), anti‐Akt [40D4] (CST), anti‐Akt (phospho Ser473) [D9W9U] (CST), anti‐p38 [D13E1] (CST), anti‐p38 (Thr180/Tyr182) [D3F9] (CST) and anti‐GAPDH [14C10] (CST). The housekeeper gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was employed as an internal control. Then the blots were incubated with secondary antibodies (anti‐rabbit or anti‐mouse, respectively). Finally, chemiluminescence was developed by ECL reagents.
5
Immunohistochemical Analysis of Tumor Markers
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The tumors were fixed in 10% formalin and embedded in paraffin followed by serial transverse sections (5 μm). The sections were deparaffinized, dehydrated, and rehydrated before IHC was performed. After blocking with 10% normal goat serum for 20 min, the sections were incubated overnight with primary antibodies at 4°C. The primary antibodies were listed as follows: anti-AFP (1:100; Affinity), anti-HEPPAR1 (1:200; Novus Biologicals), anti-JNK1 (1:100; Abcam), anti-OCT4 (1:100; Abcam), and anti-SOX2 (1:100; Abcam). Subsequently, HRP-conjugated secondary antibodies were dropped into the sections for 1 h at room temperature. The sections were then stained with the 3,3-diaminobenzidine (DAB) solution and counterstained with hematoxylin. Photographs were captured with an Olympus light microscope (Nikon, Japan).
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