Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors

The unenriched, transcribed ORFeome mRNAs were labeled with Cy5 dye

The unenriched, transcribed ORFeome mRNAs were labeled with Cy5 dye. the control-enriched library. Included in this table is the overlap of clones with a fold change 4 compared to the beads control and 1.5 compared to bead-coupled dasatinib in the presence of excess unbound dasatinib competitor. Protein kinases were significantly overrepresented within this list (DAVID Bioinformatics12, INTERPRO annotation, 0.0003). Table S4. Sequences of DNA primers used in this study. NIHMS612753-supplement-supp_tables.xlsx (81K) GUID:?4B4060C9-9D0B-462E-A272-C230710D2AF8 01: Figure S1. Gateway-compatible PLATO platform. The ORFeome library in RAF1 an entry vector is recombined into the pRD-DEST vector by Tubeimoside I an LR reaction. The DNA template containing human Tubeimoside I ORF with the T7 promoter and N-terminal TolA linker for transcription (IVT) is amplified by PCR using the common T7B (forward) and TolAk (reverse) primers.Figure S2. Preservation of mRNA enrichment for deep sequencing. To test our library mRNA recovery strategy, the untranslated and unenriched, transcribed ORFeome mRNA library was diluted 1,000 fold (input library), and p53 mRNA was spiked in at 100 times that in the input library (p53 library). (a) Product of 2nd PCR was analyzed by agarose gel electrophoresis. The DNA fragments were distributed around 400 bp. Experiments were done in duplicate (rep1 and rep2). (b) Scatter plot of clone sequencing counts of the input and p53-spiked libraries. The p53 ORF is enriched by ~100 fold in the p53 library as expected (highlighted in red). The counts of nonp53 ORFs were well correlated between the input and p53 libraries (R2 0.80). (c) RT-qPCR confirmation that enrichment of p53 is well preserved from reverse transcription to deep sequencing. The initial RT products of input and p53 libraries were subjected to RT-qPCR using primers targeting p53. The input was measured using the RDINP primer set. The relative level of p53 in the input library was set to 1 1. Data are presented as the average of two replicates. Figure S3. Analysis of LYN binding proteins. (a) ORFs ranked by enrichment on GST-LYN versus GST-MUTED. Several known and novel LYN binding candidates are highlighted in red. (b) Binding of SH2D1A and SH2D4A with LYN is phosphotyrosine dependent. GST-LYN or GST-Pep was treated with Lambda protein phosphatase prior to incubation with ribosome displayed human ORFeome. Recovered mRNA was subjected to RT-qPCR using primers targeting SH2D1A, SH2D4A and PIK3R3. Abundance was normalized to the cDNA concentration, which was measured using the RDINP primers. Fold changes were normalized to GST-Pep. Data are from = 3 experiments and presented as mean s.d. * 0.01. Figure S4. Test of PLATO using affinity-purified antibodies. Anti-p53, anti-PDCD4, and, as negative controls, anti-RBM15 and rabbit IgG (rIgG) antibodies were immobilized on protein A/G magnetic beads for immunoprecipitation of the displayed human ORFeome library. Recovered mRNA was subjected to RT-qPCR using primers targeting p53 or PDCD4. The input was measured using RDINP primer set. The relative fold of p53 or PDCD4 enrichment was normalized to rabbit IgG (rIgG). Data are from = 3 experiments and presented as mean s.d. * 0.01. Figure S5. PLATO analysis and validation of PND autoantigens. (a) Preservation of PND autoantigen enrichment from reverse transcription (RT) to deep sequencing. After immunoenrichment, one tenth of the mRNA was subjected to RT-qPCR to assess the PhIP-Seq identified autoantigens TGIF2LX and TRIM9. The fold change was normalized to rIgG. Remaining mRNA was processed for deep sequencing. Enrichment of TGIF2LX and TRIM9 was normalized to enrichment by rIgG. Data are based on = 3 experiments and presented as mean s.d. * 0.05; ** 0.01. (b) and (c) Enrichment ranking of ORF libraries IPed with Patient A and B. Several Tubeimoside I hits for patient A and B IPs are highlighted in red. (d) Heat map of selected PND patient autoantigen enrichment scores demonstrate patient specificity. Figure S6. Microarray hybridization.