The rainbow trout hepatoma cell line RTH-149 was obtained from the American Type Culture Collection (ATCC CRL-1710; Rockville, MD, USA) and cultured as previously described [33 (link)]. Escherichia coli 0111:B4 LPS and MG132 were purchased from InvivoGen (San Diego, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. The antibodies against JNK, phosphorylated JNK (Thr183/Tyr185), TAK1, phosphorylated TAK1 (Thr184/187), phosphorylated p38 MAPK (Thr180/Tyr182), K48-linkage specific polyubiquitin, K63-linkage specific polyubiquitin, HA-tag, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against p38 MAPK (ab170099) and TRAF6 (ab33915) were from Abcam (Cambridge, UK). The antibodies against 6×His tag and Flag-tag were from Invitrogen (Carlsbad, CA, USA).
K63 linkage specific polyubiquitin
Manufactured by Cell Signaling Technology
Sourced in United States
K63-linkage specific polyubiquitin is a laboratory reagent used to study ubiquitination processes. It is a polymeric form of the ubiquitin protein, specifically linked through the lysine-63 residue. This product can be used to investigate cellular signaling pathways and protein interactions involving K63-linked polyubiquitination.
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Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Phosphatase inhibitor, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
γh2ax, by Cell Signaling Technology (2 mentions)
Precast gradient gel, by Bio-Rad (2 mentions)
Ubc13, by Cell Signaling Technology (2 mentions)
Brca1, by Fortis Life Sciences (2 mentions)
Ab33915, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
K48 linkage specific polyubiquitin, by Cell Signaling Technology (1 mentions)
Mg 132, by InvivoGen (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
Ab170099, by Abcam (1 mentions)
Rth 149, by American Type Culture Collection (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
5 protocols using k63 linkage specific polyubiquitin
1
Establishing Rainbow Trout Hepatoma Cell Line
2
Antibody and Inhibitor Protocol for Protein Analysis
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Antibodies (dilutions are indicated in brackets for western blot (WB), immunofluorescence (IF), or immunoprecipitation (IP)) against FLAG (Sigma, clone M2; Sigma, produced in Rabbit, IP 3 μl/sample, IF 1:100, WB 1:1000), FLAG (Sigma, clone M2; Sigma, M, Wb 1:1000, IF 1:200), ubiquityl-histone H2A (Millipore, clone E6C5), ubiquitin (Norvus Biologicals, FK2, M, WB 1:1000, IF 1:1000; Dako WB), K48-linkage specific polyubiquitin (Enzo lifesciences, WB 1:1000), K63-linkage specific polyubiquitin (Cell Signaling, clone D7A11, 1:1000), myc (MBL, clone PL14, WB 1:3000, IF 1:100), HSC70 (Stressgen, WB 1:5000, IF 1:100), LC3B (Novus Biologicals, NB100-220), GFP (clonetech, 632381), p62 (Enzo Life Sciences, BML-PW9860), Lamin A/C (Santa Cruz, 4A58), HSC70 (Stressmarq biosciences), HSP70 (Stressgen, clone SPA-810, WB 1:1000, IF 1:50), HSPA1A (Enzo life sciences, Rb, WB 1:1000), HSPB1 (Stressmarq biosciences), GAPDH (Fitzgerald, clone 6C5, WB 1:50,000), histone H2A (Abcam, WB 1:5000), MYC (Clonetech, Mountain View, CA, USA), and DNAJB1/Hsp40 (Stressgen, San Diego, CA, USA, Rb, 1:1000) were used.
MG132 (20 µM for 3–6 h), rapamycin, Pepstatin A (10 μg/ml), E64d (10 μg/ml), 3-methyladenine (3-MA, 10 mM) ammonium chloride (NH4Cl, 20 mM) were from sigma.
MG132 (20 µM for 3–6 h), rapamycin, Pepstatin A (10 μg/ml), E64d (10 μg/ml), 3-methyladenine (3-MA, 10 mM) ammonium chloride (NH4Cl, 20 mM) were from sigma.
3
Western Blot for Ubiquitin Signaling
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Western blot analysis was performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in the RIPA buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the specific primary antibodies, followed by peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence (Denville Scientific). The following antibodies were used: antibodies to ZEB1 (1:1000, Bethyl Laboratories, A301-922A), γH2AX (1:1000, Cell Signaling Technology, 2577), K63-linkage specific polyubiquitin (1:1000, Cell signaling Technology, 12930), BRCA1 (1:1000, Bethyl Laboratories, A300-000A), Ubc13 (1:1000, Cell Signaling Technology, 4919), FLAG (1:5000, Sigma, F3165) and GAPDH (1:3000, Thermo, MA5-15738). The ImageJ program (http://rsbweb.nih.gov/ij/download.html ) was used for densitometric analysis of Western blots, and the quantification results were normalized to the loading control. Uncropped images of blots are shown in Supplementary Fig. 5 .
4
Western Blot for Ubiquitin Signaling
5
Profiling K63-linked Polyubiquitination in Adipocytes
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Immunoprecipitation was performed with differentiated adipocytes (day 8), according to the standard protocol. In brief, the cells were lysed with RIPA buffer on ice for 30 min with gentle shaking, followed by centrifugation at 12,000× g at 4°C for 12 min. The whole-cell lysate (supernatant) was then transferred to a new 1.5-ml tube. For the input sample, 20 µl whole-cell lysate was prepared with Laemmli buffer and boiled at 100°C for 5 min. For immunoprecipitation, the cell lysates were precleared for 1 h with normal rabbit IgG (Cell Signaling Technology; catalog no. 3423) and protein A/G agarose beads (Santa Cruz; catalog no. sc-2003) that had been prewashed with RIPA buffer. After the beads were removed, the precleared lysates were incubated overnight with 10 µl p62/SQSTM1 (Cell Signaling Technology; catalog no. 5114) or control rabbit IgG at 4°C with rotation. Subsequently, agarose beads were added to the lysates, incubated for another 4 h, and then collected and washed with ice-cold RIPA buffer. Immunoprecipitates were eluted from the beads by boiling in Laemmli buffer. The supernatant was loaded on a 15% SDS-PAGE gel and probed with K63 linkage–specific polyubiquitin (Cell Signaling Technology; catalog no. 5621) according to the immunoblotting protocol.
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