Quadriceps muscles were homogenized in RIPA buffer containing 1 tablet/10 ml of Complete Mini-Protease Inhibitor Mix (Roche), rotated for 2 hours at 4°C and centrifuged at 13,000g for 10 minutes at 4°C. The supernatant fraction (5 00 μg) was incubated on a rotator overnight at 4°C with 10 μl of AR antibody (Millipore, PG-21) or nonimmune rabbit IgG (Santa Cruz Biotechnology). Prewashed protein A–agarose beads (20 μl, Santa Cruz Biotechnology) were added, and samples were incubated for 1 additional hour at 4°C. Protein-antibody-bead complexes were washed 6 times in RIPA buffer in filtered spin columns (Thermo Scientific) and proteins were eluted by boiling in SDS-loading buffer for 5 minutes at 100°C. Samples were then resolved on a 7.5% SDS-PAGE gel.
Nonimmune rabbit igg
Manufactured by Santa Cruz Biotechnology
Nonimmune rabbit IgG is a purified immunoglobulin fraction from the serum of non-immunized rabbits. It is used as a control antibody in various immunoassays and other applications where a non-specific antibody is required.
Automatically generated - may contain errors
Lab products found in correlation
Non immune mouse igg, by Santa Cruz Biotechnology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Pg 21, by Santa Cruz Biotechnology (1 mentions)
Complete mini protease inhibitor mix, by Roche (1 mentions)
Anti p21, by Abcam (1 mentions)
Anti pr antibody, by Abcam (1 mentions)
Anti p27, by Abcam (1 mentions)
Pfu dna polymerase, by Promega (1 mentions)
Nprotein a sepharose, by GE Healthcare (1 mentions)
Dna restriction endonucleases, by New England Biolabs (1 mentions)
Chaps, by Merck Group (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Nf κb p65, by Merck Group (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Protein a g sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Clone 2g10, by Merck Group (1 mentions)
Epr5479, by Abcam (1 mentions)
8 protocols using nonimmune rabbit igg
1
Quantification of Androgen Receptor Protein
2
Immunoprecipitation for Phosphorylated pRB Analysis
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Immunoprecipitation assays were performed as previously described [31 (link)]. Cells were washed twice with PBS, collected and homogenized with RIPA buffer. After cell debris was removed by centrifugation, extracts were aliquoted and either used immediately or stored at -80°C. Whole-cell lysates in lysis buffer were cleared with 1.0 μg nonimmune rabbit IgG (Santa Cruz) together with 30 μl of protein A-Sepharose beads (Pierce). After centrifugation, the lysates were immunoprecipitated for 1 h at 4°C with 1 μg of the anti-Stat6 antibody or nonimmune rabbit IgG and then incubated overnight at 4°C with protein A-Sepharose. The immunoprecipitates were washed three times with lysis buffer and once with PBS and then resuspended in electrophoresis sample buffer. Samples of immunoprecipitated or total proteins (30 μg) were analyzed by Western blotting using the anti-ppRB-Ser807/811 antibody (Cell Signaling Technology) against a pRB peptide phosphorylated on the Ser807/811 residue, which is phosphorylated by both CDK2 and CDK4/6 kinases [32 ], or the anti-pRB against underphosphorylated pRB (BD Biosciences-Pharmingen), the anti-PR antibody (abcam), anti-p21(abcam), anti-p27(abcam), and anti-GADPH (as control antibody). The blots represent typical results from at least three independent experiments.
3
Immunoprecipitation and Western Blotting
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Cell culture reagents and Fluo-3 AM were obtained from Life Technologies, electrophoresis equipment and reagents from Bio-Rad, protease inhibitor cocktail (Complete) from Roche, CHAPS from Merck, nProtein-A Sepharose and glutathione Sepharose 4B from GE Healthcare, secondary antibodies from Santa Cruz Biotechnology, enhanced chemiluminescence reagents from Pierce, DNA restriction endonucleases from New England Biolabs, Pfu DNA polymerase from Promega, oligonucleotides and all other reagents were from Sigma. Antibodies used for immunoprecipitation: (1 μg) rabbit anti-HA antibody (Y-11, cat. no. sc-805, 1:40 dilution for immunoprecipitation) and non-immune rabbit IgG were from Santa Cruz Biotechnology. Antibodies used in immunoblotting were mouse anti-Myc (9E10, cat. no. sc-40, 1:500 dilution for western blotting) from Santa Cruz Biotechnology, mouse anti-HA (16B12, cat. no. MMS-101, 1:1000 dilution for western blotting) from Covance; rabbit anti-GST (1:2000) (Zissimopoulos et al., 2012 (link), 2006 (link)), and rabbit RyR2-specific (Ab1093, epitope at residues 4454-4474; 1:500) (Zissimopoulos et al., 2012 (link), 2006 (link)) antibodies.
4
SIRT1 Regulates COX-2 Transcription
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HUVECs infected with adenoviral vectors encoding SIRT1 (Ad-SIRT1) or control Ad-GFP followed by incubation of PMA (10 ng/ml) and Io (0.25 µM) for 3 h were treated with 1% formaldehyde for 15 min. The cross-linked chromatin was sheared by sonication, and the sonicated complex was used for immunoprecipitation with specific antibodies or nonimmune rabbit IgG (Santa Cruz) as control. Immunoprecipitated complexes were collected by using Dynabeads Protein A (Invitrogen). The immunoprecipitated chromatin DNA was amplified by real-time PCR using primers for COX-2 promoter region or 3′-UTR. The real-time PCR primer pairs were listed in Table S5 . Antibodies for NFATc3 (Santa Cruz) and NF-κB p65 (Millipore) were used in ChIP assay.
5
Immunoprecipitation Assay Protocol
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Cells were collected and lysed in 1 ml immunoprecipitation lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% NP-40) with complete protease inhibitor cocktail (Roche Diagnostics). The cell lysates were precleared and then incubated with the indicated antibodies for 1 h at 4 °C. The complexes were precipitated with Protein A/G-Sepharose beads (Santa Cruz Biotechnology Inc.), washed, and resuspended in 40 μl SDS loading buffer. Non-immune mouse IgG or non-immune rabbit IgG (Santa Cruz Biotechnology Inc.) served as a negative control.
6
Characterization of Phosphorylated GluA1 Antibodies
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Cantharidin (Abcam) was prepared as a 20 mM stock solution in DMSO. All other chemicals were obtained from Sigma-Aldrich. Phospho-T840 (1:2,000) and total GluA1 antibodies (1:2000, rabbit monoclonal EPR5479) were obtained from Abcam and the phospho-S845 antibody (1:1000) was obtained from Millipore. Monoclonal antibodies against a neuronal specific isoform (βIII) of tubulin (1:20,000, Clone 2G10) and AMPAR GluA2 subunits (1:1000-500, Clone N52A/42) were obtained from Sigma-Aldrich and the UC Davis/NIH NeuroMab facility, respectively. Control immunoprecipitations were performed using a nonimmune rabbit IgG obtained from Santa Cruz Biotechnology. Horseradish peroxidase conjugated secondary antibodies (1:2,000) were obtained from GE Healthcare. The phospho-T840 and phospho-S845 GluA1 antibodies used in these experiments have been extensively characterized by us (Delgado et al. 2007 (link); Gray et al. 2014 (link)) and others (Hosokawa et al. 2015 (link); Toda and Huganir 2015 (link)) and do not recognize nonphosphorylated GluA1 or GluA1 phosphorylated at other sites.
7
ChIP-qPCR Validation of NF-1 Binding
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ChIP performed as described above with sonication time reduced to 3× 10-second pulses at 10% output. Two micrograms of anti NF-1 H-300 (Santa Cruz Biotechnology) was used to ChIP 40 µg of chromatin. Equivalent amounts of nonimmune rabbit IgG (Santa Cruz Biotechnology) provided controls. Supplemental Table 2 lists quantitative polymerase chain reaction primers used to validate enrichments at specific sequences identified by ChIP sequencing (ChIP-seq).
8
Immunohistochemical Analysis of Tumor Samples
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Tumor specimens were fixed in 10 % neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and immunostained with anti-Ki67 antibody (Santa Cruz Biotechnology) and anti-CD31 antibody (abcam, San Francisco, CA, USA). Deparaffinized sections were pretreated by autoclaving in 10 % citric acid buffer (pH 8.0) at 120 °C for 15 min. Following treatment with protein block serum (Dako Cytomation, Kyoto, Japan) for 10 min and incubation with 2 % skim milk for 30 min to block nonspecific reactions, sections were incubated with primary antibody at 4 °C overnight. The EnVision polymer solution (horseradish peroxidase; Dako Cytomation) was then applied for 1 h. Signals were developed in 0.02 % 3,3′-diaminobenzidinetetrahydrochloride solution containing 0.1 %. As negative controls, the sections were incubated with tris(hydroxymethyl)aminomethane-buffered saline with non-immune mouse IgG (Santa Cruz Biotechnology) or non-immune rabbit IgG (Santa Cruz Biotechnology). Sections were then lightly counterstained with hematoxylin and examined under a fluorescence microscope (Olympus, Tokyo, Japan). For endothelial cell counting in tumors, five vascularized areas in a visual field (×100) were chosen, and the number of CD31+ cells was then counted.
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