Ubiquitylation Vs Ubiquitination

Circles represent data obtained at day; squares represent data obtained at day. CFU in the culture supernatant or released after one PBS wash were considered nonadherent and marked in deep yellow color, while CFU not released during this wash were considered adherent and marked in deep red color.All data represent the mean SD; imatinib is used at M as a nonTYKAHR inhibitor for comparison in all experiments.The percentage of perfectly matching guides of all triplicates is. The percentage of perfectly matching guides of all triplicates is. Genecentric visualization of average fold change of secondary screens in infected versus noninfected host cells.Selected type I IFN and AHRARNT pathway components are highlighted in orange and blue.Infectioninduced cytokine and chemokine production in nontargeting control, IFNAR and TYK gene knockdown THP cells.Monocytes were allowed to mature into macrophages on tissue culture treated dishes using ng ml GM CSF. When necessary, g ml kanamycin, g ml ampicillin, or g ml zeocin were added to the growth medium.. to prevent bacterial clumping. THP cells were then centrifuged, washed with PBS, and maintained in RPMI medium.Subsequently, host cells were washed and maintained for the rest of the experiments.The number of viable THP cells were counted in haemocytometer by using trypan blue. The number of viable intracellular bacteria was calculated from manually enumerated colony forming units on the agar plates.Lentiviral spinfection was performed by centrifuging well plates at, g for hours at C with THP cells grown in RPMI medium with FBS and g ml of polybrene.Twentyfour hours after lentiviral transduction, cell culture medium was replaced by RPMI with FBS and g ml of puromycin for selection.Following antibiotic selection, a library coverage of was maintained for subsequent screens.The pooled screens were performed as three independent replicates.The log fold change of each sgRNA was determined by comparing infected and uninfected samples for each biological replicate.M of primers.Relative quantification of mRNA was performed using GAPDH mRNA as internal control.Western blotting THP cells were lysed in MPER mammalian protein extraction reagent followed by shaking at C for minutes.The ratios of the uncleaved to cleaved DNA bands were used to calculate the percentage of insertiondeletion mutations in the starting cell population.Briefly, cell cultures were harvested at given time points, rapidly quenched, and spun down.Electrospray ionization was performed in negative ion mode with the following source parameters: drying gas temperature C with a flow of L min, nebulizer gas pressure psi, sheath gas temperature C with a flow of L min, capillary voltage, V, and nozzle voltage V.The extent of GFP loss was quantitated by flow cytometry.Ten days postinfection, cells were washed and resuspended in PBS supplemented with FBS.All of the statistical purchase Boc-NH-PEG3-CH2COOH details of experiments can be found in the corresponding figure legends.NGS dataset supporting the current study is available from the corresponding author upon request.Nature. Metabolomics. Immunity. Science. Tuberculosis. Color gradient of nodes represents the enrichment scores of genesets.Number of genes per category is reasch Boc-NH-PEG3-CH2COOH indicated.Correlation of hits between pooled screen and individual validation data.Diagram of eicosanoids and linoleic acid metabolism in human THP cell line.

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