HEK293 cells were plated at 3.5×106 cells/100 mm dish. Cells were transfected with 5 μg of PQCXIP AR-V7 or AR and pIRES-egfp-Vav3-myc or pIRES-Vav3-DPC-myc utilizing a calcium-phosphate transfection kit according to the manufacturer’s instructions (ClonTech). For DH interference, 22Rv1 and CWR-R1 cells were plated at 3×106 cells/100 mm dish and transfected with 10 μg DH-FLAG or EV using Lipofectamine reagent and Plus reagent (Invitrogen Life Technologies). After 48 hours, cells were lysed and immunoprecipitation was performed as previously described (36 (link)), using nonspecific mouse IgG (2 μg, Santa Cruz), monoclonal mouse anti-myc (2 μg, Invitrogen) or anti-FLAG (2 μg, Sigma), nonspecific rabbit IgG (2 μg, Santa Cruz), rabbit polyclonal anti AR-N20 (2 μg, Santa Cruz), rabbit polyclonal anti-Vav2 (2 μg, Santa Cruz), or rabbit polyclonal anti-Vav3 (2 μg, Millipore).
Nonspecific mouse igg
Manufactured by Santa Cruz Biotechnology
Nonspecific mouse IgG is a laboratory reagent used as a control in various immunological assays. It is a monoclonal antibody derived from mouse cells that does not have a specific target antigen. This product can be used to establish baseline or background signals in experiments where the presence of a target-specific antibody is being measured.
Automatically generated - may contain errors
Lab products found in correlation
Non specific rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Calcium phosphate transfection kit, by Takara Bio (1 mentions)
Rabbit polyclonal anti ar n 20, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti caveolin 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Protein a g sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
3 protocols using nonspecific mouse igg
1
Probing AR-V7 and Vav3 Interactions
2
Lipid Raft Protein Immunoprecipitation
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Equal protein
amounts of lipid rafts were incubated with 1 μg of anti-caveolin-1
antibody (#7C8) or nonspecific mouse-IgG (Santa Cruz Biotechnology)
as negative control for 16 h at 4 °C. Protein A/G-Sepharose beads
(Santa Cruz Biotechnology) were added and incubated for 1 h at 4 °C.
Beads were precipitated by centrifugation and washed four times with
PBS. Beads were boiled in SDS sample buffer for 10 min at 70 °C
before being resolved by Western blotting as described above. The
membrane was probed with anti-β-catenin (1:500), anti-galectin-1
(1:1000), and anti-caveolin-1 (#3238, 1:1000) antibodies.
amounts of lipid rafts were incubated with 1 μg of anti-caveolin-1
antibody (#7C8) or nonspecific mouse-IgG (Santa Cruz Biotechnology)
as negative control for 16 h at 4 °C. Protein A/G-Sepharose beads
(Santa Cruz Biotechnology) were added and incubated for 1 h at 4 °C.
Beads were precipitated by centrifugation and washed four times with
PBS. Beads were boiled in SDS sample buffer for 10 min at 70 °C
before being resolved by Western blotting as described above. The
membrane was probed with anti-β-catenin (1:500), anti-galectin-1
(1:1000), and anti-caveolin-1 (#3238, 1:1000) antibodies.
3
Chromatin Immunoprecipitation of C/EBP-Binding Site
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Total RNA was isolated using the RNeasy Kit (QIAGEN). For cDNA synthesis, 1 mg RNA was reverse-transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche) using Oligo(d)T primers. qRT-PCR was performed using the LightCycler 480 SYBR Green I Master Mix (Roche). Primer pairs are listed in Table S3.
Chromatin and Reporter C/EBP-Binding Site IP HEK293T cells were transfected with WT, K159/298Q-, or K159/298R-C/EBPa expression vectors for the ChIP. HEK293T cells were cotransfected with C/EBP-binding site reporter construct and WT, K159/298Q-, or K159/298R-C/EBPa-FLAG expression vectors for the C/EBP-binding site IP. ChIP assay was performed with 5 3 10 6 cells using a Bioruptor (Diagenode) for sonication (details are available on request). ChIP antibodies were against FLAG (M2, F3165; Sigma-Aldrich) and non-specific mouse IgG (Santa Cruz Biotechnology). The fold enrichment was calculated relative to the background detected with non-specific rabbit IgG. For the semi-quantitative PCR, 1/50 (1 ml) of DNA obtained from the ChIP assay was used as template in a PCR with 28 cycles. Primer pairs are listed in Table S3.
Chromatin and Reporter C/EBP-Binding Site IP HEK293T cells were transfected with WT, K159/298Q-, or K159/298R-C/EBPa expression vectors for the ChIP. HEK293T cells were cotransfected with C/EBP-binding site reporter construct and WT, K159/298Q-, or K159/298R-C/EBPa-FLAG expression vectors for the C/EBP-binding site IP. ChIP assay was performed with 5 3 10 6 cells using a Bioruptor (Diagenode) for sonication (details are available on request). ChIP antibodies were against FLAG (M2, F3165; Sigma-Aldrich) and non-specific mouse IgG (Santa Cruz Biotechnology). The fold enrichment was calculated relative to the background detected with non-specific rabbit IgG. For the semi-quantitative PCR, 1/50 (1 ml) of DNA obtained from the ChIP assay was used as template in a PCR with 28 cycles. Primer pairs are listed in Table S3.
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