Project description:Lamtor1-KO BMDCs isolated from Lamtor1flox/flox X CD11c-Cre mice were lysed by Lysis Buffer A and immunoprecipitated by anti-Lamtor1 antibody (D11H6).
Project description:To identify the specific proteins interacting with STX17, HEK293T cells stably expressing Flag-STX17 were lysed and immunoprecipitated with anti-Flag antibody, and eluted for Commassie blue staining and then subjected to mass spectrometric analysis.
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:To investigate proteins interacting with STING mutants, HEK 293T cells were co-transfected with three STING mutant expression plasmids (human STING WT, STING delCTT (aa343-379 deletion), STING del6 (aa325-379 deletion) in a pcDNA3.1+ backbone) and either pcDNA-cGAS expression plasmid or control empty pcDNA plasmid. Lysed cells were then immunoprecipitated using sheep anti-STING or control sheep anti-IgG antibodies and protein G magnetic Dynabeads.
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.
Project description:WT mice were infected with LCMV CL-13, and 100ug/ml anti-IL-17A or isotype control antibody (BioxCell) was administered on Day 0, 3 and 6. On day 12 post-infection, live CD8+ T cells were FACS-sorted from spleen according to PD-1, Tim3 and CD44 expression directly into SmartSeq low-input RNA kit lysis buffer
Project description:In an attempt to identify proteins interacting with transcription factor E2F2, proteins extracted from HA-E2F2 overexpressing HEK293T cells were were subjected to immunoprecipitation (IP) with an anti-HA antibody or, as a negative control (NegCt), with an anti-T antibody. Immunoprecipitated proteins were eluted, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The whole procedure was repeated several times in an attempt to gauge reproducibility (nine replicates of the anti-HA IP and six replicates of the control IP).
Project description:Human CD4+ Total memory cells (CD4+CD25-CD45RA-CCR7+/-) were activated for 5d with anti-CD3/anti-CD28 antibodies. About 40*10^6 cells were then irradiated twice with 254nm UV light at 0.2J and then lysed in RIPA buffer. 1mg of cell extract was incubated on a rotating wheel ~16 h at 4°C with anti-PDAP1 antibody. PDAP1:mRNA complexes were immunoprecipitated with protein-G dynabeads 4hrs at 4°C and then treated with proteinase K. RNA was then purified using TRI-reagent and RNA zymospin columns. Three independent donors were used for this experiment. Inputs were also sequenced. Total RNA was sequenced in paired-end mode. This experiment is assessing direct targets bound by PDAP1 in human T cells.
Project description:Genome-wide binding of IRF3 was analysed in MM1.S cells using ChIP-seq high-throughput approach. MM1.S cells were fixed in 1% formaldehyde (Alfa Aesar) for 15min at room temperature. cells were lysed with hypotonic lysis buffer for 15minutes followed by nuclear lysis buffer for 15min on ice. ChIP for IRF3 performed by a standard ChIP-seq protocol and the genome-wide binding of IRF3 data was analysed which revealed peaks enrichment at cell cycle genes TSS.
Project description:An RIP experiment was performed in ARP1 cell following the instructions of the article.~5-20 × 10e6 cells were harvested and lysed. Protein A/G MagBeads pre-coated with 5μg of the antibody of interest (SFRS8, abcam) and incubated with cell lysate supernatant overnight at 4°C.The beads containing immunoprecipitated RNA-protein complex were treated with 150μL of Proteinase K buffer to digest the proteins. Specific binding RNAs were isolated by using TRIzol