Dataset Information

Coimmunoprecipitation and following mass spectrometry analysis of EhRacM

ABSTRACT: To find out the binding partners of EhRacM, crosslinking and coimmunoprecipitation were performed. Then, eluted samples were submitted to Mass Spectrometry and Proteomics Core Facility, Johns Hopkins University School of Medicine and analyzed by mass spctrometry (shotgun proteomics). The coimmunoprecipitation and following analysis were conducted three times.You can find the data of HA-EhRacM (1st) from "2022_7_32_54_PM_FileSize_500998639_Byte_F010303.mzid.gz", "2022_7_32_54_PM_FileSize_500998639_Byte_F010303.mzid_2022_7_32_54_PM_File_Size__500998639__Byte__F010303.MGF", "RO-CS-LE_220912_NozakiT_MS_Rac16_WT.mzML", and "RO-CS-LE_220912_NozakiT_MS_Rac16_WT.RAW". You can find the data of pEhExHA (mock) (1st) from "2022_5_02_01_AM_FileSize_662636563_Byte_F010297.mzid.gz", "2022_5_02_01_AM_FileSize_662636563_Byte_F010297.mzid_2022_5_02_01_AM_File_Size__662636563__Byte__F010297.MGF", "RO-CS-LE_220912_NozakiT_MS_pEhExHA.mzML", and "RO-CS-LE_220912_NozakiT_MS_pEhExHA.RAW".You can find the data of HA-EhRacM (2nd) from "2022_7_35_46_AM_FileSize_794949236_Byte_F009769.mzid.gz", "2022_7_35_46_AM_FileSize_794949236_Byte_F009769.mzid_2022_7_35_46_AM_File_Size__794949236__Byte__F009769.MGF", "JS-QE-CS_221108_NozakiT_MS_S1_RA.msf", "JS-QE-CS_221108_NozakiT_MS_S1_RA.RAW", "JS-QE-CS_221108_NozakiT_MS_S1_RA.mzML". You can find the data of pEhExHA (mock) (2nd) from "2022_2_58_28_AM_FileSize_846222756_Byte_F009771.mzid.gz", "2022_2_58_28_AM_FileSize_846222756_Byte_F009771.mzid_2022_2_58_28_AM_File_Size__846222756__Byte__F009771.MGF", "JS-QE-CS_221108_NozakiT_MS_S4_RA.msf", "JS-QE-CS_221108_NozakiT_MS_S4_RA.RAW", and "JS-QE-CS_221108_NozakiT_MS_S4_RA.mzML". You can find the data of HA-EhRacM (3rd) from "2023_12_15_11_AM_FileSize_1380630557_Byte_F012916.mzid.gz", "2023_12_15_11_AM_FileSize_1380630557_Byte_F012916.mzid_2023_12_15_11_AM_File_Size__1380630557__Byte__F012916.MGF", "JS-E480-CS_230518_NozakiT_MS_S1_DDA_10pct.msf", "JS-E480-CS_230518_NozakiT_MS_S1_DDA_10pct.mzML", and "JS-E480-CS_230518_NozakiT_MS_S1_DDA_10pct.RAW". You can find the data of pEhExHA (mock) (3rd) from "2023_6_35_51_PM_FileSize_1732075728_Byte_F012915.mzid.gz", "2023_6_35_51_PM_FileSize_1732075728_Byte_F012915.mzid_2023_6_35_51_PM_File_Size__1732075728__Byte__F012915.MGF", "JS-E480-CS_230518_NozakiT_MS_S4_DDA_10pct.msf", "JS-E480-CS_230518_NozakiT_MS_S4_DDA_10pct.mzML", and "JS-E480-CS_230518_NozakiT_MS_S4_DDA_10pct.RAW".

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Entamoeba Histolytica Hm-1:imss

TISSUE(S): Trophozoite

SUBMITTER: Misato Shimoyama

LAB HEAD: Tomoyoshi Nozaki

PROVIDER: PXD042282 | Pride | 2024-06-20

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	2022_2_58_28_AM_FileSize_846222756_Byte_F009771.mzid.gz	Mzid
	2022_2_58_28_AM_FileSize_846222756_Byte_F009771.mzid_2022_2_58_28_AM_File_Size__846222756__Byte__F009771.MGF	Mzid
	2022_5_02_01_AM_FileSize_662636563_Byte_F010297.mzid.gz	Mzid
	2022_5_02_01_AM_FileSize_662636563_Byte_F010297.mzid_2022_5_02_01_AM_File_Size__662636563__Byte__F010297.MGF	Mzid
	2022_7_32_54_PM_FileSize_500998639_Byte_F010303.mzid.gz	Mzid

Items per page:

1 - 5 of 28

Similar Datasets

Project description:We have compared the response to TGFbeta1 in normal and v-rasHa transduced primary mouse keratinocytes using NCI cDNA microarrays. This analysis reveals that Ha-ras alters global TGFbeta1 mediated gene expression in a gene specific manner. The expression pattern of TGFbeta1 immediate early response genes and down regulation of cell cycle control genes is not altered by Ha-ras but the induction of most extracellular matrix genes is blocked. Using Smad3 null keratinocytes, we find that the majority of TGFbeta1 responsive genes in primary keratinocytes are Smad3 dependent, but patterns of transcriptional responses suggest that the ability of Ha-ras to block TGFbeta1 mediated gene expression is not dependent on Smad3. However, the combination of oncogenic ras and loss of Smad3 prevents the TGFbeta1 dependent suppression of genes associated with cell cycle progression and apoptosis observed with either genotype alone. In addition several extracellular matrix genes are super-repressed by TGFbeta1 in the Ha-ras Smad3 null keratinocytes. These data provide a genomic framework for understanding how disruptions of TGFbeta signaling and oncogenic ras cooperate to promote premalignant progression of primary keratinocytes. Keywords: time series design RNA from Balb/c primary and ras keratinocytes treated for 1, 6, 24, and 48 hours with TGFbeta1 at 1ng/ml was compared to RNA from the corresponding untreated controls. For each timepoint sample from primary and ras cells, multiple arrays (including dye swaps) were analyzed. There are 6, 7, 6, and 5 arrays analyzed for RNA from primary keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated primary cells, respectively. There are 6, 8, 7, and 5 arrays analyzed for RNA from ras keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated ras cells, respectively. There are 50 arrays analyzed for the Balb/c time-course dataset. RNA from Smad3 WT and KO primary and ras keratinocytes treated 48 hours with TGFbeta1 were compared to RNA from its untreated corresponding controls. There are 6 arrays analyzed for the Smad3 WT/KO data set. There are 56 arrays in this series.

Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

Dataset Information

Coimmunoprecipitation and following mass spectrometry analysis of EhRacM

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets