SPT15-myc13::HIS3 strain HQY366 (Qiu et al., 2004 (link)), isogenic to BY4741 was cultured in the presence or absence of SM treated and subjected to ChIP-seq analysis as described previously (Qiu et al., 2015 ) except that chromatin samples containing 5.0μg DNA were immunoprecipitated with anti-myc antibodies (Roche). Paired-end sequencing libraries were prepared from immunoprecipitated DNA using Illumina paired-end kits from New England Biolabs (cat. #E7370 and #E7335). Numbers of aligned paired reads from TBP-myc13 ChIP-seq and reproducibility of replicates are summarized in Table S3 . The distribution of TBP (median occupancy, and the ranges corresponding to 5-95 and 25-75 percentiles) was depicted in Fig. 5F for the 62 class “O” UC target genes in ‘O UC targets’, Data S3 .
Anti myc antibody
Manufactured by Roche
Sourced in United States
The Anti-Myc antibody is a laboratory reagent used for the detection and identification of the c-Myc protein in various biological samples. It specifically binds to the c-Myc epitope, allowing for the visualization and analysis of c-Myc expression and localization in cells and tissues.
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Lab products found in correlation
Protein g dynabead, by Thermo Fisher Scientific (4 mentions)
Complete protease inhibitor cocktail tablet, by Roche (4 mentions)
Anti flag antibody, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Dnase 1, by New England Biolabs (2 mentions)
Rnase a, by Qiagen (2 mentions)
Horseradish peroxidase, by Bio-Rad (1 mentions)
G3042, by Merck Group (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Anti rabbit horseradish peroxidase, by Merck Group (1 mentions)
Anti ha peroxidase high affinity, by Roche (1 mentions)
Alexa fluor 488 conjugated goat secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Ab16046, by Santa Cruz Biotechnology (1 mentions)
Ab16046, by Merck Group (1 mentions)
Anti his, by GenScript (1 mentions)
Protein a g plus agrose, by Santa Cruz Biotechnology (1 mentions)
Abe68, by Abcam (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 myc his, by Thermo Fisher Scientific (1 mentions)
15 protocols using anti myc antibody
1
ChIP-seq Analysis of Myc-tagged TBP
2
Immunoblot and Co-immunoprecipitation Protocols
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For immunoblot analysis, proteins were extracted from plant tissues directly into loading buffer (0.125 M Tris-HCl, pH 7.5, 4% SDS, 20% Glycerol, 0.2 M DTT, 0.02% Bromophenol Blue) while yeast protein extraction was performed following a post-alkaline method.56 (link) For co-immunoprecipitation experiments agrobacterium cultures were infiltrated at OD600 = 0.5 and proteins were extracted as previously described.57 (link) For immunodetection, proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes which were blocked in 5% skimmed milk. Membranes were incubated with Anti-HA-Peroxidase, High Affinity (Roche, ref. 12013819001) or mouse anti-GFP (Roche, ref. 11814460001) and anti-Myc antibodies (Roche, ref. 11667149001) followed by goat anti-mouse antibodies conjugated with horseradish peroxidase (Biorad, ref. 170–5047), or polyclonal rabbit anti-luciferase (Sigma-Aldrich, REF L0159), anti-GAL4BD (Sigma-Aldrich, REF G3042) and anti-VP16 (Sigma-Aldrich, REF V4388) antibodies followed by anti-rabbit horseradish peroxidase (Sigma) to detect -HA, YFP, Myc, nLUC, cLUC, GAL4BD and VP16 tagged proteins respectively. Signals were detected using the SuperSignal West Femto chemiluminescence kit (Pierce). Ponceau S was used to stain membranes to confirm equal protein loading.
3
Coimmunoprecipitation of Myc-tagged Proteins
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Coimmunoprecipitation analyses were performed as previously described in Bermudez-Lopez et al. (2015) (link). Cells were mechanically broken in 50 mM HEPES, 150 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and 0.5% Triton X-100 (pH 7.5) supplemented with Complete protease inhibitor cocktail tablets (from Roche). Cell extracts were incubated with beads for 2 h at 4°C. Myc-tagged proteins were immunoprecipitated using anti-Myc antibodies (Roche, 9E10) coupled to protein G Dynabeads (Invitrogen). Bound proteins were eluted with SDS-PAGE loading buffer and analyzed by Western blot.
4
CHIP Dimerization and E2 Binding Assay
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For the E2 association assay, cells transfected with CHIP WT, CHIP p.Glu278fs, or CHIP Δ278–303 were harvested in NP40 lysis buffer (50 mM Tris–HCL pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NA3VO4, 0.1 M NaF, 0.1% NP40, 1 mM PMSF). Cell lysates (1 mg) were incubated overnight with anti-FLAG antibodies (3.8–4.2 μg, Sigma-Aldrich, USA) at 4 °C. For the dimerization assay, cells transfected with CHIP-Myc along with CHIP WT-FLAG, CHIP p.Glu278fs-FLAG, CHIP Δ278–303-FLAG were harvested in NP40 lysis buffer. Cell lysates (1 mg) were incubated overnight with anti-FLAG antibodies (3.8–4.2 μg, Sigma-Aldrich, USA) or anti-Myc antibodies (2 μg, Roche, Switzerland) at 4 °C. Immunocomplexes were isolated with protein A-Sepharose beads saturated with 1% bovine serum albumin (BSA) by rotating for 5 h at 4 °C. After washing, bound proteins were denatured, eluted, and resolved by 12% polyacrylamide SDS-PAGE.
5
Isolation of Myc-tagged Protein Complexes
6
Myc-tagged Protein Purification
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One-hundred and twenty OD600 of asynchronous cells were OD600 = 1 were collected and washed in cold water and resuspended in 200 μL of ice-cold buffer A (50 mM HEPES, 150 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and 0.5% Triton X-100 [pH 7.5] supplemented with complete protease inhibitor cocktail tablets, Roche). Five-hundred mL of glass beads (425–600 μm) were added and cells lysed in a FastPrep FP120 cell disruptor (BIO101) by three repetitions of a 20 s cycle at power setting 5.5. Extracts were maintained on ice for 2 min after each cycle. Cell extracts were centrifuged for 10 min at 12,000 r.p.m. at 4°C and the supernatant incubated with protein G Dynabeads (Invitrogen) bound to anti-Myc antibody (Roche, 9E10) for 2 hr at 4°C. Finally, beads were washed five times in washing buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5% Triton) and unbound from the antibody by incubating at 37°C for 4 min in SR buffer (2% SDS, 0.125 M Tris-Cl, pH 6.8). Immunoprecipitated proteins were mixed with SS buffer (5% saccharose, 0.0125% bromophenol blue) and run in an SDS-PAGE gel.
7
Immunofluorescence Staining of Myc-GLUT1 Expressing Cells
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Immunofluorescence (IF) staining of cells was performed as previously described [32 ]. In brief, cells cultured on coverslips were washed with ice-cold PBS and fixed with methanol. Cells were permeabilized with PBS containing 0.2% Triton X-100. The cells expressing Myc-GLUT1 were incubated with anti-myc antibody (9E10, Roche) overnight followed by Alexa Fluor® 488-conjugated goat secondary antibody (Invitrogen) for 1 h. The coverslips were mounted in Vectashield (Vector Laboratories) and examined by a confocal laser-scanning microscope.
8
Co-Immunoprecipitation Assay Protocol
9
Rab39b Protein Expression in Cell Lines
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Two myc-tagged constructs encoding either wild type or mutant (p.G192R) RAB39B protein were created using the vector pcDNA 3.1/myc-His (Invitrogen Life Technologies, Carlsbad, CA). Rat pheochromocytoma (PC12) and human neuroblastoma (SK-N-BE(2)C) cells were grown and transfected with wild type or mutant constructs using either a retrovirus (pCL Vector System, Orbigen, San Diego, CA) for PC12 cells or Lipofectamine (Invitrogen) for SK-N-BE(2)C cells using previously described methods [21 (link)]. After 24 h the cells were lysed, and the lysates were subjected to 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). The membranes were incubated with an anti-myc antibody (Roche Life Sciences, Branford, CT) and detection was achieved using the Novex ECL chemiluminescent substrate reagent kit (Invitrogen).
10
Assessing cIAP1/2 Binding to RIP1
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To assess the impact of compounds on cIAP1 and cIAP2 binding to RIP1, 700,000 HEK293T cells were seeded into 100 mm cell-culture plates and transfected the next day with 6 μg of plasmid DNA, myc-RIP1 in pRK5 vector. After 24 h, cells were lysed in NP-40 buffer (20 mM Tris-HCl [pH 7.5], 135 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol) supplemented with EDTA-free protease inhibitors (Roche) and 1 mM DTT. Lysates from 6 plates were pooled and divided into equal 300 μL aliquots, into which 7 μg of purified GST-fusion proteins (GST-cIAP1-BIR3 or GST-cIAP2-BIR3) were added in the presence of various concentrations of SMAC mimetic compounds. The tubes were rocked overnight at 4°C, and the next day 20 μL of glutathione-Sepharose beads (GE Healthcare) was added to each tube and the tubes were rocked for an additional 2 h at 4°C. The GST-fusion proteins were recovered by centrifugation at 2,300 g for 30 sec, then washed 3-times in NP-40 lysis buffer. Finally, 20 μL of 2X Laemmli buffer was added and the samples were boiled, then fractionated by SDS-PAGE, followed by immunoblotting using anti-myc antibody (Roche) for detection of myc-RIP1 and anti-GST antibody (BD Biosciences, La Jolla, CA) for detection of GST-fusion proteins.
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