HeLa and HCC94 cells transfected with NC-miR or miR-195-5p mimics were seeded into 24-well plates (5.0 × 105 cells/well). After 24 h, the cells were fixed in 4% paraformaldehyde for 10 min (at room temperature), permeabilized with 0.05% Triton X-100 and blocked with 5% normal goat serum (Beyotime, Shanghai, China) for 1 h (at room temperature). Next, the cells were incubated with primary antibody of MAP3K8 (Abcam, ab137589, concentration 1: 100, MA, USA) for 18 h at 4°C. Followed by that, the cells were washed three times with PBS, incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077; 1:200; MA, USA) for 2 h at 37°C and stained with DAPI (Beyotime, Shanghai, China) at room temperature for 15 min. For detecting MAP3K8 expression in the tumor tissues, the tumor tissues were fixed in 4% paraformaldehyde and 4-μm sections were also prepared. After being permeabilized with 0.05% Triton X-100, blocked with 5% normal goat serum, the sections were, respectively, incubated with primary antibody of MAP3K8 (Abcam, ab137589, concentration 1: 100, MA, USA) and goat anti-rabbit IgG H&L (Alexa Fluor® 594) (ab150080; 1:200; MA, USA). DAPI (Beyotime, Shanghai, China) was used for staining nucleus. The immunofluorescence signal was observed using a fluorescence microscope (×200, Olympus Company).
Ab137589
Manufactured by Abcam
Sourced in United States
Ab137589 is a laboratory equipment product. It serves a core function in scientific research and experimentation. No further details can be provided in a concise, unbiased, and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal ecl, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Beyotime (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Goat anti rabbit igg h l alexa fluor 594, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab214362, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab128915, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
4 protocols using ab137589
1
Immunofluorescence Assay of MAP3K8 Expression
2
Western Blot Analysis of Signaling Proteins
3
Immunohistochemical Analysis of MEK1/2 and MAP3K8
4
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The tissues or cells were collected and lysed with RIPA lysis solution (Beyotime Biotechnology, Shanghai, China) and the total protein was extracted. Next, the protein (30 μg) underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred to polyvinylidene fluoride (PVDF) membranes, sealed with 5% skimmed milk powder, and incubated with primary antibodies at 4°C overnight. On the next morning, the membranes were washed with TBST buffer and incubated with horseradish peroxidase (HRP) -labeled anti-rabbit secondary antibodies (Abcam, ab6721, concentration 1: 1,000, MA, USA) for 1 h at room temperature. After washing the membranes by TBST, ECL was added for brands exposure, and Image-J was applied to analyze OD value. Primary antibodies used in this study were as follows: E-cadherin (Abcam, ab40772, concentration 1: 1,000, MA, USA), Snail (Abcam, ab18203, concentration 1:1,000, MA, USA), Vimentin (Abcam, ab92547, concentration 1: 1,000, MA, USA), MAP3K8 (Abcam, ab137589, concentration 1: 1,000, MA, USA), p-ERK (Abcam, ab214362, concentration 1: 1,000, MA, USA), ERK (Abcam, ab17932, concentration 1: 1,000, MA, USA), GAPDH (Abcam, ab128915, concentration 1: 2,000, MA, USA).
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