For coimmunoprecipitation, cells were lysed in SDS-free RIPA (0 mM Tris, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 1.0% Triton X-100, pH 7.4) supplemented with PMSF and a protease inhibitor cocktail on ice for 10 min, followed by centrifugation at 13 000g for 20 min. Protein (20 μg, “input”) was removed and lysate was added to Protein G Dynabeads (Invitrogen: 10003D) preincubated with c-Myc antibody (Santa Cruz sc-40). After 12 h of enrichment, beads were washed (3×, SDS-free RIPA) and total protein eluted with 50 mM glycine, “output.” Both input and output protein were subjected to separation by SDS-PAGE. After SDS—PAGE, proteins were transferred onto methanol-preactivated Immobilon-P PVDF membranes (pore size 0.45 μm; Millipore) using a semidry transfer cell. After transfer, membranes were treated in accordance with standard Western blotting procedures, using a solution of 3% BSA (ThermoFisher) in TBST (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). Membranes were probed with GABARAP (Abcam), c-Myc (Santa Cruz), and calnexin (Abcam) antibodies overnight. Following secondary antibody incubation, membranes were visualized using SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher) and recorded on a chemiluminescent Western blot imaging system (Azure Biosystems C300).
Gabarap
Manufactured by Abcam
Sourced in United Kingdom
GABARAP is a protein that plays a role in the attachment of the cytoskeleton to GABA receptors, which are important for neurotransmission. It is involved in the regulation of GABA receptor trafficking and function.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
C myc antibody, by Santa Cruz Biotechnology (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Calnexin, by Abcam (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Phosphorylase inhibitor cocktail, by Roche (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
P mtor, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Mmp14, by Abcam (1 mentions)
P akt, by Bioworld Technology (1 mentions)
P p70s6k, by Proteintech (1 mentions)
P ikkβ, by Cell Signaling Technology (1 mentions)
Mek1 2, by Cell Signaling Technology (1 mentions)
6 protocols using gabarap
1
Co-immunoprecipitation and Western Blot
2
Western Blot Analysis of Cellular Signaling Pathways
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Cells were lysed using RIPA lysis buffer (Solarbio) containing protease inhibitors (Beyotime) and a phosphorylase inhibitor cocktail (Roche) to obtain protein, and the contents were 990 μl RIPA lysis buffer + 10 μl PMSF + 100 μl phosphorylase inhibitor. A BCA Protein Assay Kit was used to confirm the concentrations of protein separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% BSA blocking reagent for 1 h at room temperature (RT) and incubated with primary antibodies overnight at 4° C. The membranes were washed and incubated for 1 h at RT with secondary antibodies. The proteins were analyzed using the ECL Plus kit. The following antibodies used included at a dilution of 1:1000: GABARAP (Abcam, ab109364), E-cadherin (Abcam, ab40772), N-cadherin (Abcam, ab76011), vimentin (Proteintech, 10366-1-AP), MMP2 (Abcam, ab110186), MMP14 (Abcam, ab3644), AKT (Abcam, ab179463), p-AKT (Bioworld Technology, Ser473, BS4007), mTOR (Abcam, ab2732), p-mTOR (Ser2448; 5536), p70S6K (Proteintech, 14485-1-AP), and p-p70S6K (Thr389; 9234), MEK1/2 (8727), p-MEK1/2 (Ser217/221; 9154), ERK1/2 (4695), p-ERK1/2 (Thr202/Tyr204: 4370), IKK-β (8943), p-IKK-β (2078), IκBα (4812), and p-IκBα (2859) from Cell Signaling Technology. β-actin was used as an internal control (ZSGB-BIO, TA-09, dilution 1:1500).
3
Immunoblotting Analysis of GAD Autoantibodies
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Human GAD65 (Diamyd, Stockholm, Sweden), GAD67 (Abnova, Taipei, Taiwan), and GABARAP (Abnova) recombinant proteins were electrophoretically separated and transferred to a polyvinylidene difluoride membrane and strips incubated with the patient's serum (1:1000 dilution), GAD65 (GAD-6, Hybridoma Bank, Iowa City, IA), GAD67 (Abcam, Cambridge, UK), or GABARAP (Abcam) commercial antibodies followed by biotinylated goat antihuman IgG or horse antimouse IgG (Vector Laboratories, Burlingame, CA) and developed with diaminobenzidine tetrahyrochloride [15 (link)]. In some experiments we used an enhanced chimioluminiscence technique (ECL Western Blotting System) following manufacturer’s instructions. The specificity of GAD65 and GAD67 commercial antibodies was confirmed with immunoblots with recombinant proteins and also with HEK293 cells (see below) expressing the corresponding antigens.
4
Western Blot Analysis of Autophagy Markers
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Frozen cell pellets were lysed using RIPA lysis buffer (Santa-Cruz, ref: SC24948) plus complete protease inhibitor cocktail (Roche, ref: 11836153001). The amount of proteins in cell lysates was determined by BCA protein assay (Thermo Scientific, ref: 23225), and 10 µg of protein were loaded on 10% NuPAGE Bis-Tris gel (Invitrogen) for separation and transferred to PVDF membrane (BioRad). BOLT and NUPAGE Bis-Tris 4–12% gradient gels (Invitrogen) were used for separation of pro-LC3B and LC3B-II. Membranes were blocked in 2% milk and incubated in primary antibodies overnight at 4 °C. Primary antibodies were diluted in 2% milk and included: p62 (1:1000; Sigma, ref: P0067), LC3B (1:1000; Abcam, ref: ab48394), GABARAP (1:1000; Abcam, ref: ab109364), beta-actin (1:10000; Abcam, ref: ab6276), and vinculin (1:1000; Abcam, ref: ab129002). Membranes were then washed with 1X PBS-T (0.1%), incubated in HRP-conjugated secondary antibodies and detected using Bio-Rad Clarity Western ECL substrate and the electronic Bio-Rad ChemiDoc MP System. Densitometry was performed using Image Lab software (Bio-Rad), and the relative levels of LC3B-II were determined by first normalizing to the actin loading controls then to the DMSO controls.
5
Western Blot Analysis of Autophagy Markers
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Twenty mg of frozen muscle tissue from 3 PM, 3 sIBM, 1 DM, 2 JDM, and 4 controls were lysed in radioimmuneprecipitation assay buffer (50 mmol/L Tris-HCl, pH 8.0, 1% Nonidet P-40, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA), freshly supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 1 mmol/L phenylmethanesulfonyl fluoride (Sigma-Aldrich). Protein extracts (40 µg) were separated by NuPAGE Novex 4–12% Bis-Tris protein gels (Invitrogen, Life Technologies), and transferred to a nitrocellulose membrane. After blocking, samples were incubated with antibodies to autophagy-related ubiquitin-like modifier LC3 B (Sigma-Aldrich) and γ-aminobutyric acid type A receptor associated protein (GABARAP) (Abcam), followed by appropriate secondary antibodies. Bound antibodies were visualized by enhanced chemiluminescence with ECL Plus Substrate (Pierce, Thermo Fisher Scientific).
6
Protein Aggregation and Solubility Analysis
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Protein samples were brought to 10 mM Tris, pH 6.8, 1 mM EDTA, 40 mM DTT, 1% SDS, 10% sucrose by addition of 5 × sample buffer boiled 5 min and loaded onto 4–15% pre-cast criterion SDS-PAGE gradient gels (Bio-Rad). To analyze protein aggregation and solubility, fractions were extracted with buffers of increasing solubilizing strength, using first low salt, then non-ionic detergents, ionic detergents and urea as previously described [17 (link)]. Antibodies used include TTBK1 kinase domain (Clone F28701.1 from [22 (link)]), TTBK1 C-terminal domain (ProScience #5013), Actin (clone AC40, Sigma A4700), and GABARAP (Abcam ab109364). Secondary goat anti-mouse or goat anti-rabbit IgG were the secondary antibody reagents used at a dilution of 1:1000 (GE Lifesciences). Immunoblot quantitation was performed using Image J software.
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