For western blot: anti-USP5 (abcam, ab154170, 1:1000), anti-SLUG (Affinity, DF6202, 1:1000), N-cadherin (abcam, ab18203, 1:1000), Occlaudin-3 (Affinity, AF0129, 1:1000) Cytokeratin (Affinity, BF0197, 1:1000), myosin (Affinity, AF4725, 1:1000). For IF: anti-E-cadherin (CST, 14472, 1:50), anti-Vimentin (CST, 5741, 1:100), anti-SLUG (Affinity, DF6202, 1:100) and anti-USP5 antibodies (Abcam, ab154170, 1:100). For IHC: anti-E-cadherin (CST, 14472, 1:100), anti-Vimentin (CST, 5741,1:100), anti-SLUG (CST, 9585, 1:100), anti-USP5 (abcam, ab154170, 1:50), anti-Ki67 (Abcam, ab15580, 1:500).
Ab154170
Manufactured by Abcam
Sourced in United States
Ab154170 is a laboratory equipment product. It is a core tool designed for specific scientific applications. The product's function is to facilitate the performance of fundamental research procedures in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Df6202, by Abcam (3 mentions)
Ab15580, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
Ab154170, by Affinity Biosciences (1 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions)
N storm, by Nikon (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab109201, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
Ab124957, by Abcam (1 mentions)
Ab2787, by Abcam (1 mentions)
Ab32041, by Abcam (1 mentions)
Ab27969, by Abcam (1 mentions)
Ab76055, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab178872, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Ab16502, by Abcam (1 mentions)
7 protocols using ab154170
1
Epithelial-Mesenchymal Transition Protein Analysis
2
Immunohistochemical Analysis of EMT Markers
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The tissues were deparaffinized with xylene and dehydrated with ethanol of decreasing concentrations. Endogenous peroxidase was blocked by incubating with 3% hydrogen peroxide for 15 min. The antigen retrieval was done in a steam pressure cooker with citrate buffered saline (pH 6.0) for 15 min at 95 °C. After incubation with normal goat serum for 20 min at room temperature to block unspecific labeling, the tissues were incubated with primary antibodies including anti-E-cadherin (CST, 14472, 1:100,), anti-Vimentin (CST, 5741, 1:100,), anti-SLUG (CST, 9585, 1:100), anti-USP5 (abcam, ab154170, 1:50), anti-Ki67 (Abcam, ab15580 1:500) antibodies in a humidified chamber overnight at 4 °C. Diaminobenzidine was utilized for color development and hematoxylin as counterstain. Expression levels of E-cadherin, Vimentin, USP5, and SLUG were independently evaluated by two investigators.
3
Formononetin Probe Staining of SLUG and USP5
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PLC-PRF-5 cells treated with or without Formononetin probe were fixed by 3.7% formaldehyde and blocked with 5% FBS containing 0.1% TritonX-100. Samples were then stained with anti-SLUG (Affinity, DF6202, 1:100) and anti-USP5 antibodies (Abcam, ab154170, 1:100) and secondary antibodies coupled to AlexaFluor 488 or 594 (Invitrogen). Formononetin probe was connected to 647 dyes by click reaction. Pictures were taken with the N-stochastic optical reconstruction microscopy system (N-STORM, Nikon, Tokyo, Japan).
4
Protein Expression Analysis of Cellular Pathways
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After lysing in RIPA lysis buffer, the collected total protein was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%), shifted onto polyvinylidene fluoride membranes, and then blocked by 5% nonfat milk. The membranes were probed all night at 4 °C with the primary antibodies against loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA) and p65 (ab16502, 1/1000; Abcam), Histone H3 (ab1791, 1/1000; Abcam), IKKα (ab32041, 1/10,000; Abcam), IKKβ (ab124957, 1/1000; Abcam), NEMO (ab178872, 1/5000; Abcam), p-IKBα(Ser36) (ab133462, 1/10,000; Abcam), p-IKBβ(Ser23) (Cat# 4921 S, 1/1000; Cell Signaling Technology, Danvers, MA, USA), CD9 (ab92726, 1/2000; Abcam), CD63 (ab134045, 1/1000; Abcam), CD81 (ab109201, 1/1000; Abcam), HSP70 (ab2787, 1/1000; Abcam), E-cadherin (ab76055, 1/1000; Abcam), α-SMA (Cat# 19245S, 1/1000; Cell Signaling Technology), TGF-β1 (ab27969, 1/2000; Abcam), Collagen type I (ab34710, 1/1000; Abcam), Collagen type III (ab7778, 1/5000; Abcam), Usp5 (ab154170, 1/1000; Abcam). Next, membranes were washed in TBS-T, and then incubated for 2 h with horseradish peroxidase-labeled secondary antibodies at room temperature. Electrochemiluminescence luminous liquid was employed for analyzing protein bands as instructed by supplier (Pierce, Rockford, IL, USA). Results were visualized after developing in the dark.
5
Immunofluorescence Assay for Cell Markers
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For the immunofluorescence assay, PLC-PRF-5 and Hep3B cells subjected to different treatments were washed three times with 1 × PBS, fixed in 4% paraformaldehyde (pre-cooled at 4 °C, Solarbio) for 20 min, and blocked with 5% bovine serum albumin (BSA, KeyGEN BioTECH) containing 0.1% TritonX-100 (Sigma) for 30 min at room temperature. Then, the resultants were incubated with anti-E-cadherin (CST, 14472, 1:50) and anti-Vimentin (CST, 5741, 1:100) or anti-SLUG (Affinity, DF6202, 1:100) and anti-USP5 antibodies (Abcam, ab154170, 1:100). After washing with 1 × PBS again, and then incubated with fluorescently conjugated secondary antibodies (1:200, KeyGEN BioTECH) diluted in 5% BSA for about 50 min at room temperature. Finally, the cells were washed with 1 × PBS and mounted with the DAPI-containing mounting medium (Solarbio). The images of cells were taken with a laser scanning confocal microscope (Leica).
6
Western Blot Analysis of Protein Expression
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Firstly, RIPA lysis buffer (Beyotime Biotechnology, China) was utilized to extract total proteins. Protein concentration was then measured using BCA™ Protein Assay Kit (Pierce, Appleton, USA). Subsequently, protein extracts were separated using 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk for 2 h at room temperature, and then incubated overnight at 4 °C with diluted primary antibody. Following washed with TBST, the membranes were incubated with secondary antibody for 2 h at room temperature. Finally, the ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester, NY) was utilized to detect target bands. The primary antibodies used were as follows: anti-PAI1 (13801-1-AP, Proteintech, USA), anti-SERBP1 (14088-1-AP, Proteintech), anti-SIX5 (abcam, UK), anti-GAPDH (60004-1-Ig, Proteintech), anti-USP5 (ab154170, Abcam, Cambridge, MA, USA) and anti-Histone 3 (ab1791, Abcam, Cambridge). The original western blots were shown in Supplemental Material.
7
Molecular Mechanisms of Cell Cycle Regulation
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The human GBM cell lines U251 and DBTRG-05MG, and human embryonic kidney cell line (293T) were purchased from the American Type Culture Collection. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml of penicillin and 100 units/ml of streptomycin. The primary antibodies against USP5 (ab154170), CyclinD1 (ab134175), CyclinE1 (ab33911), CDK2 (ab32147), CDK4 (ab108357), Ki67 (ab16667), PCNA (ab18197), Ubiquitin (linkage-specific K48, ab140601) were purchased from Abcam (USA), CDK6 (13,331), and β-Actin (3,700) were purchased from Cell Signal Technology (USA). MG-132 (S2619) was purchased from Selleckchem (USA). Doxycycline hyclate (DOX, D9891) and Cycloheximide (CHX,239763) were purchased from Sigma (USA).
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