Total lysate was extracted from the indicated cells in RIPA buffer and incubated with anti-USP33 (Abcam), anti-c-Myc (Abcam), or control IgG (Santa Cruz) at 4°C. After 2 h, b protein A/G Plus agarose beads (Santa Cruz) was added and incubated at 4°C for another 2 h. Following washing four times with RIPA buffer, the protein complexes were mixed with sample buffer, boiled for 5 min, separated by SDS-PAGE, and finally subjected to western blot analysis with anti-USP33, anti-c-Myc, or anti-ubiquitin (Abcam).
Anti c myc
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany
Anti-c-Myc is a primary antibody that recognizes the c-Myc protein, a transcription factor involved in cell growth and proliferation. It can be used for applications such as Western blotting, immunoprecipitation, and immunohistochemistry to detect and study the c-Myc protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin d1, by Abcam (30 mentions)
Anti β catenin, by Abcam (23 mentions)
Anti gapdh, by Abcam (21 mentions)
Pvdf membrane, by Merck Group (16 mentions)
Anti gapdh, by Cell Signaling Technology (11 mentions)
Anti β actin, by Abcam (9 mentions)
Anti e cadherin, by Abcam (8 mentions)
Anti vimentin, by Abcam (8 mentions)
Anti n cadherin, by Abcam (7 mentions)
Anti β catenin, by Cell Signaling Technology (7 mentions)
Anti bcl 2, by Abcam (7 mentions)
Anti β actin, by Merck Group (7 mentions)
Anti ki67, by Abcam (6 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Anti flag, by Merck Group (5 mentions)
Anti cdk4, by Abcam (5 mentions)
Anti e cadherin, by Cell Signaling Technology (5 mentions)
Anti bax, by Abcam (5 mentions)
Anti gsk3β, by Abcam (5 mentions)
157 protocols using anti c myc
1
Immunoprecipitation and Western Blot Analysis
2
Hepatocellular Carcinoma Tissue Microarray Analysis
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For IHC, HCC tissue microarrays analysis (TMA) slides were purchased from Alenabio (Xi'an, China). The primary antibodies used in IHC were anti-p-p38 (Cell signaling technology (CST), Boston, MA, USA, #4511), anti-p38 (Abcam, Hongkong, China, #ab31828), anti-p-MKK3/6 (CST, #9236), anti-MKK3 (Epitomics, Burlingame, CA, USA, #1728), anti-MKK6 (Epitomics, #1640), anti-YAP (CST, #12395), anti-c-Jun (CST, #2315), anti-c-Myc (Epitomics, #1472), anti-ki67 (Abcam, #ab15580), anti-AFP (Abcam, #ab46799) and anti-SIRT1 (for mSIRT1, CST, #2028; for hSIRT1, Abcam, #ab32441). For IF, the primary antibodies used were anti-p-p38 (for mp-p38, CST, #4511; for h-p-p38, Abcam, #ab45381), anti-p38 (Abcam, #ab31828), anti-p-MKK3/6 (CST, #9236) and anti-FLAG-tag (CST, #8146). For WB, the primary antibodies used were anti-GAPDH (CST, #5174), anti-p-p38 (CST, #4511), anti-p38 (Abcam, #ab31828), anti-SIRT1 (for mSIRT1, CST, #2028; for hSIRT1, Abcam, #ab32441), anti-p-MKK3/6 (CST, #9236), anti-MKK3 (Epitomics, #1728), anti-MKK6 (Epitomics, #1640), anti-FLAG-tag (CST, #8146), anti-AFP (Abcam, #ab46799) and anti-YAP (CST, #12395). All IHC, IF and WB experiments were performed conventionally and the protocols are available elsewhere.
3
Western blot analysis of protein expression
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The cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed by lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton 100 and 0.1% SDS; pH 8.0) for 20 min on ice. Protein concentrations were determined by Bradford assay. Equal amounts of proteins were subjected to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline and Tween 20 (TBST; 50 mM Tris, 150 mM NaCl and 0.1% Tween-20; pH 7.5) containing 5% skimmed dry milk for 2 h at room temperature, and then incubated with primary anti-RbAp48 rabbit monoclonal (Epitomics, Inc., Burlingame, CA, USA), anti-c-Myc (Epitomics, Inc.), anti-GATA-1 rabbit monoclonal (CST, Boston, MA, USA) or anti-β-actin rabbit monoclonal (Sigma-Aldrich, St. Louis, MO, USA) antibodies overnight at 4°C. The membranes were then washed and incubated with goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase antibody (Sigma-Aldrich) for 2 h at room temperature. The proteins were visualized by chemiluminescence using an enhanced chemiluminescence kit (Advansta, Inc., Menlo Park, CA, USA). Immunoblots were quantified by Quantity One software (Bio-Rad, Hercules, CA, USA).
4
Investigating Wnt/β-catenin Pathway Regulation
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Rhein was purchased from MUST Biotech (Chengdu, China). LiCl was purchased from Sigma‐Aldrich (St. Louis, MO, USA). MG132 was from Merk Millipore Corporation (Darmstadt, Germany). Cycloheximide (CHX) was from Beyotime Biotechnology (Jiangsu, China). Anti‐β‐catenin, anti‐c‐Myc, anti‐PCNA, anti‐phosphorylated GSK‐3β (Ser9) antibodies were from Epitomics (Burlingame, CA, USA). Anti‐GSK3 antibody was from Signal way Antibody LLC (College Park, MD, USA). Anti‐cyclin‐D1 antibody was from Proteintech (Rosemont, IL, USA). The plasmid for phosphorylation‐deficient mutant of β‐catenin (S33A, S37A, T41A, S45A) was from Addgene (Cambridge, MA, USA).
5
Protein Expression Profiling by IHC and WB
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IHC and Western blotting were performed as described previously [11 (link)]. The primary antibodies included those used for immunofluorescence studies as well as anti-Cyclin-D1 and anti-c-Myc (both from Epitomics, Burlingame, CA, USA). β-actin was used as a loading control.
6
Immunoprecipitation and Western Blotting
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IP and western blotting were performed as our previous report.24 (link) Briefly, cells were transfected, treated with 10 μM MG132 for 6 h, and lysed using 2 × RIPA buffer (Tris-HCl, pH 7.4 (100 mM), NaCl (300 mM), 1% NP-40, 2% sodium deoxycholate, 10 mM NaF, and 10 mM Na vanadate). The cell lysates were cleared by centrifugation and incubated with 1 μg antibody for 1 h at 4 °C followed by incubation with 15 μl protein A and G beads (Santa Cruz, Santa Cruz, CA, USA) for 2 h at 4 °C. Immunoprecipitates were subjected to western blotting. For western blotting analysis, cells were scraped from the dishes into the lysis buffer. A total of 25 mg of total protein was separated by SDS-PAGE and blotted with anti-c-Myc (Epitomics, Burlingame, CA, USA), anti-NPM (Epitomics), anti-EBP2 (Abnova, Taipei City, Taiwan, China), anti-β-catenin (CST, Danvers, MA, USA), anti-IκB-α (CST), anti-α-tubulin (Abmart, Shanghai, China), anti-actin (Abmart), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abmart).
7
Investigating p63 Regulation in Cancer Cells
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H1299 or HeLa cells with 50% confluency in 6-well plates were co-transfected with 400 ng pCMV2-FLAG-MM1, 200 ng pEGFP and 400 ng pcDNA3.1/pcDNA3.1-ΔNp63α/pcDNA3.1-ΔNp63γ with 2.5 μl lipofectamine 2000 (Invitrogen). 24 hours later, cells were collected, washed with phosphate-buffered saline, and resuspended in EBC250 lysis buffer (50 mM Tris-HCl, pH8.0, 250 mM NaCl, 0.5% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 50 mM NaF, and 0.5 mM Na3VO4). Protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad). An equal amount of protein (about 50 μg total protein) was loaded, separated on a 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and hybridized to an appropriate primary antibody and horseradish peroxidase-conjugated secondary antibody for subsequent detection with ECL (Millipore). IB analysis was performed with anti-p63 (Santa Cruz, sc-8431, 1:200), anti-FLAG (Sigma, F1804, 1:1000), anti-MM1 (Epitomics, 5689-1, 1:500), anti-c-Myc (Epitomics, 1472-1, 1:1000), anti-GFP (Santa Cruz, sc-9996, 1:1000), anti-p21 (Abcam, ab54562, 1:500), anti-Cyclin D1 (Epitomics, 2261-1, 1:1000), anti-CDK4 (Santa Cruz, sc-260, 1:1000), or anti-Actin (Abcam, ab13772, 1:1000).
8
Immunostaining of Cellular and Tissue Samples
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Cells and tissue samples were fixed (4% PFA), quenched (0.1 M glycine, 120 mM phosphate buffer, pH 7.4, 30 min, 4 °C), and permeabilized in blocking buffer (0.4% Saponin for cell cultures, 3% Triton X-100 for brain slices; 120 mM phosphate buffer, 1% BSA, pH 7.4, 1 h, 4 °C). Samples were then incubated with primary antibody (1:100–1:300 v/v in blocking buffer; 1–2 h at 23 °C or O/N at 4 °C) followed by secondary antibody (1:200 v/v in blocking buffer, 1–2 h at 23 °C). Samples were washed with blocking buffer (4 °C), rinsed in PBS, and mounted (Fluorsave, EMD Millipore). Primary antibodies used: anti-PSD95 (mouse IgG2a, monoclonal; clone 7E3-1B8, Merck Millipore), anti-c-Myc (mouse monoclonal clone 9E10; Abcam, Cambridge, UK), anti-GAD 65 (mouse IgG2a, monoclonal; clone GAD-6; Sigma), anti-p38 synaptophysin48 (link) (mouse IgG1, monoclonal; clone 7.2; Synaptic Systems, Goettingen, Germany), anti-GluR-1 (rabbit polyclonal; generated in house), anti-SNAP25 (rabbit polyclonal; cat. 111002, Synaptic Systems), anti-p65 synaptotagmin (rabbit and goat polyclonal46 (link)), anti-GFP (rabbit polyclonal; cat. A6455, Thermo Fisher Scientific). Secondary antibodies used: Donkey anti-Mouse, anti-Rabbit, and anti-Goat AlexaFluor488/568/647 (Jackson ImmunoResearch).
9
Western Blot Analysis of Protein Samples
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Proteins were separated by SDS-PAGE using NuPAGE™ 4-12% (w/v) Bis-Tris gradient gels (ThermoFisher Scientific Inc., Waltham, MA, USA). For Western detection, proteins were transferred onto a PVDF membrane using the iBlot™ 2 dry blotting system (ThermoFisher Scientific Inc., Waltham, MA, USA). The membrane was blocked in 3% (w/v) bovine serum albumin (BSA) dissolved in 1x PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.05% (v/v) Tween 20). Western blots were reacted with anti-HapX antisera (1:10,000; Davids Biotechnologie, Regensburg, Germany) or with polyclonal anti-c-Myc (1:1000; ab9106 Abcam plc, Cambridge, UK) as primary antibodies and with monoclonal anti-rabbit IgG peroxidase (1:10,000; A1949 Merck KGaA, Darmstadt, Germany) as secondary antibody. The membrane was developed using the 1-Step™ Ultra TMB-Blotting chromogenic substrate (ThermoFisher Scientific Inc., Waltham, MA, USA).
10
Western Blot Analysis of Tumor Proteins
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The cell and xenograft tumors were lysed and liquid supernatant was collected. The protein concentration was determined using a bicinchoninic acid assay (Beyotime, Haimen, China). The sample proteins were electrophoresed on 12% and 8% sodium dodecyl sulfate-polyacrylamide gel (Beyotime) and transferred to a polyvinylidene fluoride membrane (Beyotime) for 60 min at 100 V. Next, the membranes were blocked with 5% skim milk at room temperature and then incubated and shaken overnight at 4°C with mouse anti-CITED1 (1:250, Abcam, Boston, MA, USA), anti-actin (1:5000, Proteintech, Wuhan, China), and rabbit anti-beta-catenin (1:5000, Abcam), anti-c-myc (1:1000, Abcam), anti-cyclinD1 (1:1000, Abcam). After washing, the membranes were treated with goat anti-mouse antibody (1:10000, MultiSciences, Hangzhou, China) and goat anti-rabbit antibody (1:4000, Proteintech) at room temperature for 1 h. Membranes were visualized by chemiluminescence (Thermo Fisher Scientific, New York, NY, USA) using the ECL-advance Western blotting Detection System (ChemiDocXRS+, Bio-Rad). Actin was used as the endogenous control.
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