Both HA-tagged and Flag-tagged full length Twist and Slug were cloned into pcDNA3.0. In addition, full length Twist and Slug were cloned into pEGFP-N1. The WT Dub3 construct was purchased from Addgene. CS Dub3 was generated using the QuikChange Mutagenesis kit (Stratagene, La Jolla, CA, USA) as described previously [47 (link)]. All sequences were verified by DNA sequencing. The antibodies used include: anti-Flag, anti-Actin, anti-Myc (Sigma-Aldrich, St. Louis, MO, USA), anti-Dub3 (Abcam), anti-Ub (Millipore), N-cadherin (Upstate), anti-Slug, anti-Twist (Cell Signaling), anti-Vimentin, anti-ERα (Neomarkers), anti-HA (Roche), anti-E-cadherin, anti-Claudin-7 (BD Bioscience). Dub3 shRNA expression plasmids were purchased from MISSION shRNA at Sigma-Aldrich (St. Louis, MO, USA). WP1130 and PR619 were from Selleck. Smartpool siRNA against human Dub3 was from Dharmacon (Chicago, IL, USA).
Anti ub
Manufactured by Merck Group
Sourced in United States
Anti-Ub is a lab equipment product developed by Merck Group. It is designed to detect and isolate ubiquitinated proteins, which are proteins that have been modified by the attachment of a ubiquitin molecule. The core function of Anti-Ub is to facilitate the study of protein ubiquitination, a cellular process involved in various biological pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti myc, by Merck Group (3 mentions)
Anti flag, by Merck Group (3 mentions)
Wp1130, by Selleck Chemicals (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti dub3, by Abcam (2 mentions)
Pr 619, by Selleck Chemicals (2 mentions)
Anti ha, by Roche (2 mentions)
Mission shrna, by Merck Group (2 mentions)
Anti e cadherin, by BD (2 mentions)
Anti ha, by Merck Group (2 mentions)
Anti twist, by Cell Signaling Technology (1 mentions)
Anti slug, by Cell Signaling Technology (1 mentions)
Anti snail1, by Cell Signaling Technology (1 mentions)
Claudin7, by Abcam (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti tuj1, by Merck Group (1 mentions)
Anti notch3, by Santa Cruz Biotechnology (1 mentions)
Anti cc3, by Merck Group (1 mentions)
Hrp conjugated donkey anti goat igg, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated goat anti mouse or anti rabbit igg, by Enzo Life Sciences (1 mentions)
7 protocols using anti ub
1
Genetic Constructs and Antibodies for Twist and Slug Regulation
2
Generation and Validation of Dub3 Mutants
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Plasmids of wild-type and deletion mutants for Snail1 were generated as described26 (link). The WT-Dub3 was from addgene. Dub3 (C89S) was generated using the QuikChange Mutagenesis kit (Stratagene, La Jolla, CA) as described previously31 (link). All sequences were verified by DNA sequencing. Deletion mutants of Dub3 were constructed as described previously31 (link). Antibodies used include: anti-Flag (F3165, 1:4,000, anti-Actin (A2228, 1:10,000), anti-Myc (9E10, 1:3,000) from Sigma-Aldrich (St. Louis, MO), Anti-Dub3 (Abcam, ab12991, 1:1,000); anti-Ub (Millipore, MAB1510, 1:500), N-cadherin (Upstate, 05-915, 1:1,000), anti-Snail1 (Cell Signaling, 4719, 1:1,000), Vimentin (Ab-2, 1:2,000) and ERα (Ab-15, 1:1,000) from Neomarkers, anti-HA (Roche, 3F10, 1:10,000), and anti-E-cadherin (610181, 1:10,000, BD Bioscience) and Claudin-7 (Abcam, ab27487, 1:1,000). Dub3 shRNA expression plasmids were purchased from MISSION shRNA at Sigma-Aldrich (St. Louis, MO). WP1130 and PR619 were from Selleck. Smartpool siRNA against human Dub3 was from Dharmacon (Chicago, IL).
3
Immunoblot Analysis of Cellular Proteins
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Immunoblot analysis was carried out as previously described with slight modifications12 (link). Briefly, cell lysates were prepared in immunoprecipitation (IP) buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% NP-40, 1% sodium deoxycholate with 1 mM PMSF, 1 μg/μl aprotinin, and 1 μg/μl leupeptin as protease inhibitors) and incubated on ice for 30 min. Total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with anti-Tuj1 (1:1,000, Millipore), anti-Ub (1:1,000, Millipore), anti-Notch3 (1:200, Santa Cruz Biotechnology), anti-CC3 (1:1,000, Millipore) or anti-β-Actin antibody (1:2,000, Santa Cruz Biotechnology). Based on the types of primary antibodies, the appropriate HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000, Enzo Life Sciences) or HRP-conjugated donkey anti-goat IgG (1:5,000, Santa Cruz Biotechnology) was used.
4
Investigating Protein Regulation in HEK293 Cells
5
Isolation and Immunoblot Analysis of Apple Proteins
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Protein isolation from apple fruit and calli and immunoblot analysis were performed as previously described (Li et al. 2015 (link)). The protein concentration of each extract was measured using a BCA protein assay kit (Cat. no. P0012S, CWBIO). Purified recombinant MdNAC72 was used to generate a specific antibody in rabbit (Abmart, China, http://ab-mart.com.cn/ ). Anti-His (Cat. no. HT501; TransGen Biotech, China), anti-GST (Cat. no. HT601; TransGen Biotech), anti-MBP (Cat. no. HT701; TransGen Biotech, Beijing, China), anti-GFP (Cat. no. HT801; TransGen Biotech, Beijing, China), anti-FLAG (Cat. no. 14793S; Cell Signaling Technology, USA), anti-Myc (Cat. no. HT101; TransGen Biotech), anti-Ub (Cat. no. ST1200, Sigma, USA), anti-phospho-p44/42 (Cat. no. 4730; Cell Signaling Technology), anti-phosphoSer/Thr (Cat. no. ab117253, Abcam, UK), and anti-tubulin (Cat. no. M20045, Abmart) antibodies were diluted 1:1,000 with Tris-buffered saline with Tween 20 (TBST buffer, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% [v/v] Tween 20) and incubated with nitrocellulose membranes (Cat. no. S80209, Pall Corporation, USA). Secondary antibodies (goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated; Cat. no. HS201 or HS101, TransGen Biotech) were diluted to 1:3,000 in TBST.
6
Western Blotting of Protein Complexes
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Cells were lysed in SDS loading buffer. The boiled samples were separated by 10% SDS–PAGE and transferred to nitrocellulose (NC) membranes (Whatman, GE Healthcare). The membranes were blocked with 5% milk and incubated with different antibodies overnight at 4, followed by incubation with secondary antibodies. The primary antibodies used in western blotting included anti-Flag M2, anti-HA, anti-Ub, anti-Dlg5 (Sigma-aldrich); anti-β-TrCP (Cell signaling) and anti-Cullin1, anti-SKP1 and anti-β-actin (Santa Cruz).
7
Proteomic Analysis of VPS23A Interactome
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The IP assay was conducted with transgenic plants that overexpressed Myc/FLAG-tagged versions of VPS23A. Total proteins were extracted from 10-day-old transgenic seedlings with native buffer (50 mM Tris-MES [pH 8.0], 0.5 M sucrose, 1 mM MgCl 2 , 10 mM EDTA) supplemented with 5 mM DTT, 1 mM PMSF, protease inhibitor cocktail cOmplete Mini tablets (one per 10 ml) (Roche) and 0.2% NP40. The supernatants were collected after centrifugation at 12 000 g at 4 C three times for 5 min each time. Then, the 26S proteasome inhibitor MG132 was added to the sample extracts of Myc-VPS23A, and they were incubated with anti-Myc/Flag magnetic beads (ChromoTek) or p62-agarose (Enzo Life Sciences) at 4 C for 2 h. After washing three times with PBS buffer (pH 7.4), the bead-bound proteins were eluted in sodium dodecyl sulfate (SDS) sample buffer. The sample was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) for 10 min. For ubiquitination analysis, western blots were performed with anti-Myc (Easy Bio), anti-FLAG (Sigma), anti-Ub (stock from our own laboratory), anti-K48-linkage-specific polyubiquitin chains (anti-K48-Ub, Cell Signaling Technology), and anti-K63-linkagespecific polyubiquitin chains (anti-K63-Ub, Cell Signaling Technology) antibodies. For LC-MS/MS analysis, the gel was stained with Coomassie brilliant blue to obtain the candidate interacting proteins of VPS23A.
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