Project description:DZIP (DAZ-Interacting Protein) containing a C2H2 zinc-finger domain is expressed predominantly in human embryonic stem cells and fetal and adult germ cells; DZIP colocalizes with DAZ and/or DAZL proteins in these tissues. DZIP may associate with DAZ and its other cofactors in an RNA-binding protein complex that functions in both embryonic stem cells and germ cells. To identify mRNAs associated with human DZIP1 protein in HeLa cells, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip).DZIP1 ribonucleoprotein (RNP) complexes were performed with 2 µg of anti-DZIP1 antibody (rabbit polyclonal, Santa Cruz Biotechnology, CA, USA) bound to protein A-agarose beads (Sigma, Deisenhofen, Germany). HeLa cells were lysed in polysome lysis buffer (Tris-HCl pH 7,4 15mM, MgCl2 15 mM, NaCl 0,3 M, 1% Triton X-100, 1 mM DTT, 100 U / ml RNase Out, PMSF 1mM and E64 10uM) for one hour at 4°C. Beads were washed, then buffer and cell lysate were added, and the reaction mixtures were tumbled for 2 hours at 4°C. After this incubation, the beads were thoroughly washed again with polysome lysis buffer and then either RNA extracted for microarray and RT-PCR experiments using the RNeasy mini kit (Qiagen). To control for non-specifically enriched RNAs, identical IPs were performed with beads precoated with rabbit IgG as a negative control. RNA was processed for hybridization with GeneChip 3? IVT Express (Affymetrix - Santa Clara, USA), according to the manufacturers instruction. Briefly, cDNA was synthesized from immunoprecipitated RNA using reverse transcriptase followed by second strand synthesis to generate double-stranded cDNA. An in vitro transcription reaction was used to generate biotinylated cRNA. After purification and fragmentation, cRNA was hybridized onto GeneChip Affymetrix Human Genome U133 Plus 2.0 arrays. Post hybridization washes were preformed on an Affymetrix GeneChip® Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip® Scanner 3000. Scanned arrays were normalized using GCRMA in Partek software (Partek Incorporated. St. Louis, MO). Differentially enriched RNAs were found after performing One-way ANOVA analysis comparing immunoprecipitated samples against control samples. Final lists of genes were obtained by filtering the data from the statistical results according to fold enrichment more than 2 and false discovery rate (FDR 5%).

Project description:DZIP (DAZ-Interacting Protein) containing a C2H2 zinc-finger domain is expressed predominantly in human embryonic stem cells and fetal and adult germ cells; DZIP colocalizes with DAZ and/or DAZL proteins in these tissues. DZIP may associate with DAZ and its other cofactors in an RNA-binding protein complex that functions in both embryonic stem cells and germ cells. To identify mRNAs associated with human DZIP1 protein in HeLa cells, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip).DZIP1 ribonucleoprotein (RNP) complexes were performed with 2 µg of anti-DZIP1 antibody (rabbit polyclonal, Santa Cruz Biotechnology, CA, USA) bound to protein A-agarose beads (Sigma, Deisenhofen, Germany). HeLa cells were lysed in polysome lysis buffer (Tris-HCl pH 7,4 15mM, MgCl2 15 mM, NaCl 0,3 M, 1% Triton X-100, 1 mM DTT, 100 U / ml RNase Out, PMSF 1mM and E64 10uM) for one hour at 4°C. Beads were washed, then buffer and cell lysate were added, and the reaction mixtures were tumbled for 2 hours at 4°C. After this incubation, the beads were thoroughly washed again with polysome lysis buffer and then either RNA extracted for microarray and RT-PCR experiments using the RNeasy mini kit (Qiagen). To control for non-specifically enriched RNAs, identical IPs were performed with beads precoated with rabbit IgG as a negative control. RNA was processed for hybridization with GeneChip 3? IVT Express (Affymetrix - Santa Clara, USA), according to the manufacturers instruction. Briefly, cDNA was synthesized from immunoprecipitated RNA using reverse transcriptase followed by second strand synthesis to generate double-stranded cDNA. An in vitro transcription reaction was used to generate biotinylated cRNA. After purification and fragmentation, cRNA was hybridized onto GeneChip Affymetrix Human Genome U133 Plus 2.0 arrays. Post hybridization washes were preformed on an Affymetrix GeneChip® Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip® Scanner 3000. Scanned arrays were normalized using GCRMA in Partek software (Partek Incorporated. St. Louis, MO). Differentially enriched RNAs were found after performing One-way ANOVA analysis comparing immunoprecipitated samples against control samples. Final lists of genes were obtained by filtering the data from the statistical results according to fold enrichment more than 2 and false discovery rate (FDR 5%). DZIP IP and control IP samples were analyzed for each of 3 technical replicates.

			Action	DRS
	11_30S013_Sample02.msf	Msf
	11_30S013_Sample02.raw	Raw
	result%2030-S-013.sf3	Other

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Lamtor1-interacting proteins in Lamtor1-KO BMDCs

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