The following commercial antibodies were used in this study; monoclonal antibodies to GFP (Abcam, ab1218), LC3 (Cosmo Bio, CTB‐LC3‐2‐1C), ubiquitin (clone FK2; Enzo); polyclonal antibodies to Salmonella (Abcam), GFP (Life Technologies).
Clone fk2
Manufactured by Enzo Life Sciences
Sourced in United States
The Clone FK2 is a laboratory product manufactured by Enzo Life Sciences. It is a recombinant protein that serves as a detection reagent for the identification of poly-ubiquitinated proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti atg5, by Cell Signaling Technology (1 mentions)
Rabbit anti bcl 2, by Cell Signaling Technology (1 mentions)
Ab5847, by Merck Group (1 mentions)
Anti ha clone c29f4, by Cell Signaling Technology (1 mentions)
Anti ubiquitin clone p4d1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin clone c4, by Merck Group (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Merck Group (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Fluorescent secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti phospho histone h3 ser10, by Merck Group (1 mentions)
5 protocols using clone fk2
1
Antibody Detection for Salmonella and GFP
2
Antibody Characterization for Cellular Analysis
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The antibodies used and their sources are as follows: mouse anti–β-actin (622101; BioLegend); rabbit anti–βIII-tubulin (802001; BioLegend); mouse anti-ubiquitin (MMS-258R; Covance; or clone FK2; Enzo Life Sciences); mouse anti–HA-tag (MMS-101R; Covance); mouse anti–β-tubulin (clone E7; DSHB); rabbit anti–neurofilament M (AB1987; EMD Millipore); mouse anti–MYC-tag (clone 9E10; DSHB); mouse anti–FLAG-tag (clone M2; Sigma-Aldrich); rabbit anti-GFP (598; MBL); rabbit anti-FBXW7 (ab109617; Abcam); rabbit anti-HUWE1/MULE (sc-49768; Santa Cruz Biotechnology, Inc.); rabbit anti-BECLIN1 (PD017; MBL); rabbit anti-VPS34 (GTX129528; GeneTex); rabbit anti-BCL2 (50E3; Cell Signaling Technology); rabbit anti-MCL1 (ABIN126984; antibodies-online.com); rabbit anti–phospho-MCL1 (SAB4504259; Sigma-Aldrich); mouse anti-prohibitin (Clone II-14-10; Thermo Fisher Scientific); rabbit anti-p62 (PM045; MBL); rabbit anti-ATG5 (2630; Cell Signaling Technology); and rabbit ATG7 (2631; Cell Signaling Technology). HRP-conjugated (Vector Laboratories), Alexa Fluor 565–conjugated, and Alexa Fluor 488–conjugated (Molecular Probes) antibodies were used as secondary antibodies for detection.
3
Characterization of GluA1 Receptor Mutants
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The plasmids encoding full-length wild-type pH-GluA1 and the K868R and qKR mutants have been described previously (23 ). The pH-sensitive GFP (pHluorin) tag was inserted after the signal peptide sequence in the extracellular N-terminal region of GluA1. C-terminal serine to alanine or aspartate mutants for pH-GluA1 were generated using the standard overlap extension polymerase chain reaction protocol.
Specific antibodies against GluA1 (4.9D; 1:5,000) and GFP (JH4030; 1:1,000) were generated in the Huganir laboratory and have been characterized previously (23 ). The following antibodies were purchased from commercial sources: anti-ubiquitin clone P4D1 (Santa Cruz Biotechnology; 1:1,000) and clone FK2 (Enzo Life Sciences), anti-GluA1 phospho-Ser-831 (Millipore, AB5847; 1:2,500), anti-GluA1 phospho-Ser-845 (Millipore, AB5849; 1:2,500), anti-HA clone C29F4 (Cell Signaling Technology; 1:1,000), anti-synaptophysin clone 7.2 (Synaptic System; 1:10,000), and anti-β-actin clone C4 (Millipore; 1:10,000).
Specific antibodies against GluA1 (4.9D; 1:5,000) and GFP (JH4030; 1:1,000) were generated in the Huganir laboratory and have been characterized previously (23 ). The following antibodies were purchased from commercial sources: anti-ubiquitin clone P4D1 (Santa Cruz Biotechnology; 1:1,000) and clone FK2 (Enzo Life Sciences), anti-GluA1 phospho-Ser-831 (Millipore, AB5847; 1:2,500), anti-GluA1 phospho-Ser-845 (Millipore, AB5849; 1:2,500), anti-HA clone C29F4 (Cell Signaling Technology; 1:1,000), anti-synaptophysin clone 7.2 (Synaptic System; 1:10,000), and anti-β-actin clone C4 (Millipore; 1:10,000).
4
Western Blot Analysis of Protein Expression
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Cells were lysed in lysis buffer (1.5% SDS, 1% NP-40, 10 mM Tris, pH 8.0, and 1 mM EDTA) containing a protease inhibitor cocktail (cOmplete™, Roche). Sonication was performed and the sample was then boiled at 95°C for 6 min. Total protein concentrations were measured using BCA assay (Sigma–Aldrich). Protein samples were separated by SDS/PAGE and transferred on to PVDF membranes. After blocking with 5% skim milk in TBS containing 0.1% Tween 20, membranes were incubated with antibodies specific for desmoglein1 (Dsg1), desmoglein3 (Dsg3), tubulin, actin (Santa Cruz Biotechnology, Santa Cruz, CA), poly(ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Danvers, MA, U.S.A.), ubiquitinylated conjugates (FK2 clone, Enzo Life Sciences, lnc., Farmingdale, NY, U.S.A.), GAPDH, or Dsp (Thermo Fisher Scientific). Membranes were washed using TBS with 0.1% Tween-20 and incubated with secondary antibodies. Signals were detected using an ECL solution (Supersignal West Pico, Thermo Fisher Scientific).
5
Immunofluorescence Staining Protocol
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Commercial primary antibodies and dilutions were used as following: mouse anti-mono- and polyubiquitinylated proteins, 1:100 (FK2 clone, Enzo Cat. No. BML-PW8810); rabbit antiphospho-Histone H3 Ser10, 1:1000 (EMD Millipore Cat. No. 06-570); mouse anti-Rpn3/PSMD3 (G-1), 1:500 (Santa Cruz Biotecnology Inc Cat. No. sc-393588); Rabbit anti-β-galactosidase (Cappel MP Biomedicals, 1:500); mouse anti-Armadillo (DSHB, 1:100), mouse anti-Prospero (DSHB, 1:100) and mouse anti-Dacapo (DSHB, 1:4). Rat anti-Delta antibody, 1:500, was a gift from M. Rand (University of Rochester, USA). Rabbit anti-Atg8a, 1:200, was a gift from G. Juhasz (Eotvos Lorand University, Hungary). Fluorescent secondary antibodies were from Jackson Immunoresearch.
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