Journal: Developmental cell

Article Title: YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis in Vivo

doi: 10.1016/j.devcel.2019.01.017

Figure Lengend Snippet: (A) Gene tracks with enhancers (green). Control regions closer to promoter than enhancer used for 3C (yellow). Promoters (purple). C:P, Control to Promoter contact. E:P, Enhancer to Promoter contact. (B) Cartoon of difference between 2D and 3D enhancer-promoter proximity. (I) 2D proximity. (II) 3D proximity (III) fixing DNA/chromatin interactions and digesting DNA. Loci not in contact diffuse away. (IV) Ligation of loci in contact. (V) qPCR of ligated loci. E:P interactions are more common than C:P, reflecting contact frequency (C) Quantification of chromatin contact in CM nuclei by 3C at genomic loci indicated in Fig. 7A, normalized to Control CM enhancer-promoter contacts. Groups compared: ANOVA with post-hoc Bonferroni tests. Data shown mean +/− SEM. n=3 replicates/group (from pooled CM nuclei from 6 Control hearts or 6 YAP5SA OE hearts, STAR methods). Controls: both YAP5SA no tam and tam-injected MCM control. See Table S2 for primers.

Article Snippet: T4 DNA ligase (3C) , NEB , Cat#R3575L.

Techniques: Ligation, Injection

Journal: Developmental cell

Article Title: YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis in Vivo

doi: 10.1016/j.devcel.2019.01.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: T4 DNA ligase (3C) , NEB , Cat#R3575L.

Techniques: Plasmid Preparation, Recombinant, Imaging, SYBR Green Assay, Expressing, Software, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, In-silico folding predictions for circRNA architectures pre- and post-ribozyme excision. Predictions were run using RNAfold and the colors indicate positional entropy values. Parent architectures do not contain MS2 and are the designs published by Litke and Jaffrey which correspond to addgene plasmids #124360 (left) and #124362 (right). b, MS2-circRNAs containing barcodes and MS2 hairpins. MS2-circRNA 1b contains an added priming site specifically for reverse transcription. c, In-gel DFHBI-1T fluorescence of total RNA extracted from cells expressing MS2-circRNAs is b, each expressed using 2-3 barcode sequences of varying GC content. Gels 1, 4, and 5 were non-denaturing 6% acrylamide gels and gels 2 and 3 were denaturing 10% acrylamide gels. d, Structure (PDB ID: 2BU1) of the MCP dimer bound to an MS2 hairpin (top). Linear representations of monomeric and tandem-dimer MCP protein fusions (bottom). In a tandem dimer, the N-terminus of one MCP protomer is linked to the adjacent C-terminus of a second MCP protomer by a short linker. e, Variants of MCP-Lck protein fusions were co-expressed with MS2-circRNA 2. Lck was enriched using dasatinib-conjugated agarose beads and the co-enriched MS2-circRNA was visualized by in-gel DFHBI-1T fluorescence. DMSO or free dasatinib was included during enrichment to probe for specific protein enrichment. td-indicates tandem dimer designs. VE/AG variants contain V75E and A81G mutations which prevent MCP oligomerization. delFG variants possess internal deletions (V67-A81) of the FG loop which also prevent MCP oligomerization. All MCP variants contain the high affinity V29I mutation. The linkers are positioned between MCP protomers. The delFG variant was used in all subsequent experiments and is referred to as ‘tdMCP’ throughout the manuscript.

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: In Silico, Reverse Transcription, Fluorescence, Expressing, Protein Enrichment, Mutagenesis, Variant Assay

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, Subassembly, used to link protein variants to nucleotide barcodes, is achieved by sequencing the cloned plasmid library using long-read high-throughput sequencing. In parallel, the plasmid library is integrated into the RPLP to generate a cell line stably expressing tdMCP:MS2-circRNA complexes for use in biochemical experiments. b, Schematic of the RPLP cell line. Recombination of Bxb1 attB-containing plasmids into the RPLP stably integrates protein variants and MS2-circRNA barcodes under genomic transcriptional control. Selection for successful recombinants is run using AP1903 and/or puromycin. c, Parallel Flag immunoprecipitations were performed using serial dilutions of lysate generated from RPLP cells stably expressing Flag-tdMCP-Braf WT and a library of 20,000 MS2-circRNA 1b barcode sequences. Pearson’s R correlation coefficients of barcode sequence counts are reported for replicate enrichments or RT-PCR amplifications. d, Sequenced barcode counts derived from two independent expressions and Flag immunoprecipitations of Flag-tdMCP-BTK co-expressed with a library of MS2-circRNA 2 barcode sequences. Pearson’s R = 0.98. e, Schematics of MS2-circRNA 1b, the architecture used to encode variants in the tdMCP- and untagged BRaf variant libraries, and dummy MS2-circRNA which is included in the lysis buffer to minimize barcode sequence missassociation. f, Relative frequencies of barcode sequence counts matched to Myc-tdMCP-BRaf and Flag-tdMCP-BRaf libraries after 45 min Myc immunoprecipitation. g, Quantification of the data shown in f . The y-axis is the fraction of barcode sequence counts encoding the Flag-tagged library (n=2 expression, lysis, enrichment, and sequencing replicates).

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Next-Generation Sequencing, Stable Transfection, Expressing, Control, Selection, Generated, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Variant Assay, Lysis, Immunoprecipitation

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, Sequenced barcode counts derived from two independent Flag immunoprecipitations (IP) of Flag-tdMCP-BRaf co-expressed with a library of MS2-circRNA 1b barcode sequences (Pearson’s R = 0.93). b, Experimental workflow of the barcode sequence misassociation experiment. Dummy MS2-circRNA does not contain a barcode or the priming site required for reverse transcription. c, Results from the experiment shown in b . Relative frequencies of barcode sequence counts matched to Flag-tdMCP-BRaf and Myc-tdMCP-BRaf libraries in unenriched lysate (left) and after 105 min Flag immunoprecipitation performed with lysate that was generated with dummy MS2-circRNA in the lysis buffer (right). d, Quantification of the data shown in c . The y-axis is the fraction of barcode sequence counts encoding the Myc-tagged library (n=2 expression, lysis, enrichment, and sequencing replicates).

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Derivative Assay, Sequencing, Reverse Transcription, Immunoprecipitation, Generated, Lysis, Expressing

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, Western blot analysis of HEK293 RPLP cells stably expressing tdMCP-BRaf variants. b, AlphaFold2 model (AF-15056-F1) of full-length human BRaf. Varied positions in the untagged and tdMCP-BRaf variant libraries are shown as orange spheres. c, Schematic depicting the LABEL-seq abundance assay in which a tdMCP standard (Myc-tdMCP-SNAP) is co-expressed with a tdMCP-variant library. Abundance scores are WT-normalized ratios of variant barcode sequence counts derived from parallel Flag and Myc immunoprecipitations (IP). d, Comparison of abundance measurements obtained by western blotting (right, y-axis = ratio of Flag signal to Myc signal normalized to Flag-tdMCP-BRaf WT-expressing cells treated with DMSO) or by quantifying co-enriched MS2-circRNA barcode sequences with high-throughput sequencing. (left, y-axis = the ratio of barcode sequence counts derived from parallel Flag and Myc enrichments normalized to Flag-tdMCP-BRaf WT-expressing cells treated with DMSO). HEK293T cells co-expressing MS2-circRNAs, the Myc-tdMCP-SNAP standard, and Flag-tdMCP-BRaf WT (pink circles) or G446V (blue triangles) were treated with DMSO or the variant-selective degrader SJF0628 for 24 h prior to lysis. The average of 4 barcode sequence ratios per condition is shown per replicate (marked by symbol). Error bars = standard error of the mean (SEM) of replicate scores. Westerns are shown in Extended Data 3f. e, Sequence-abundance map for BRaf variants in the tdMCP-BRaf library. The abundance scores shown are the average of two replicates, each involving an independent Myc-tdMCP-SNAP standard transduction, cell lysis, parallel Flag and Myc immunoprecipitations, and quantification by high-throughput sequencing. Black dots in the map indicate the WT amino acid and gray tiles indicate missing data. Position-averaged abundance scores are shown in the bar graph above the heatmap. f, LABEL-seq abundance score distributions for all synonymous WT and nonsynonymous BRaf variants. The gray dashed line indicates the abundance score value (>2 standard deviations of the synonymous distribution lower than WT) we defined as decreased abundance. g, Individually assessed variant abundances measured by western blotting (n=3) compared to abundance scores determined with the LABEL-seq abundance assay for a panel of BRaf variants (Pearson’s R = 0.95). Error bars = SEM. h, Position-averaged abundance scores for each position in the tdMCP-BRaf variant library (represented as spheres) projected onto the structures of (left) autoinhibited (PDB ID: 6NYB) and (right) active BRaf (PDB ID: 4MNE). The shade of blue indicates position-averaged abundance score: white = 1, darkest blue = 0.72.

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Western Blot, Stable Transfection, Expressing, Variant Assay, Sequencing, Derivative Assay, Comparison, Next-Generation Sequencing, Lysis, Transduction

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, Western blot analysis of HEK293T cells expressing the pErk reporter and treated with EGF (50 ng/mL, 10 min). Based on quantitative enrichment of pErk, it appears only a small fraction of the pErk reporter is phosphorylated under this regime. b, Scatterplot showing activity score correlations between two independent activity assays run with overexpressed CRaf. Scores from each assay correspond to the average of two replicates, each involving independent cell culturing, lysis, parallel pErk and Flag immunoprecipitations, and quantification with high-throughput sequencing (Pearson’s R = 0.74). c, Activity scores (with overexpressed CRaf) for previously characterized activating BRaf variants. Mean barcode score +/- SEM for each replicate included in b. d, LABEL-ses activity score distributions for all synonymous WT and nonsynonymous BRaf protein variants. The gray dashed lines indicate activity score values (>2 standard deviations of the synonymous WT distribution higher or lower than WT) we defined as activating and inactivating. e, Activity scores (with CRaf) for BRaf variants classified as activating (32.8%), Wild Type-like (64.9%), or inactivating (2.3%). f, Activity scores (with CRaf) for every BRaf variant at each position. g, Positions with the lowest 10 median activity scores shown as blue spheres on an active BRaf complex (PDB ID: 6Q0K). Positions 26, 189, and 443 are not resolved in the structure. h, The autoinhibited BRaf complex (PDB ID: 6NYB) with BRaf’s CRD shown in blue, non-CRD BRaf positions shown in black, and 14-3-3 shown in green. G258 is positioned within the CRD, which plays a role in autoinhibition, Ras binding, and membrane localization . In autoinhibited BRaf, the phi-psi angles at position G258 are only energetically accessible for glycine suggesting variants at this position alter the backbone conformation and may disrupt autoinhibitory contacts, thereby leading to activation. i, Autoinhibited BRaf (PDB ID: 6NYB) with BRaf P367 shown as red sticks, non-P367 BRaf positions shown in blue and 14-3-3 in green. P367 is positioned in the CRD-kinase domain linker which binds 14-3-3 in the autoinhibited BRaf complex. Based on the known binding specificity of 14-3-3 proteins , we speculate that P367 variants lead to BRaf activation by disrupting autoinhibitory 14-3-3 interactions.

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Western Blot, Expressing, Activity Assay, Cell Culture, Lysis, Next-Generation Sequencing, Variant Assay, Binding Assay, Membrane, Activation Assay

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, General schematic of the LABEL-seq activity assay. b, The canonical Raf-Mek-Erk signaling pathway. c, Activities (represented as the ratio of pErk and Flag barcode sequence counts) of BRaf WT and V600E in HEK293T cells co-expressing Flag-tdMCP-Erk2 (pErk reporter) and MS2-circRNAs. Cells were lysed, parallel Flag and pErk immunoprecipitations were performed, and the ratio of pErk/Flag counts for each co-enriched MS2-circRNA barcode sequence was calculated. Center line = mean; dotted lines = inner quartiles. n=10-11 barcodes per variant. ****P<0.001. d, Schematic of the LABEL-seq assay for the multiplexed measurement of untagged BRaf variant activity with the pErk reporter. e, Sequence-activity map for variants in the untagged BRaf library co-expressed with CRaf. The activity scores shown are the average of two replicates, each involving independent cell culturing, lysis, parallel pErk and Flag immunoprecipitations, and quantification with high-throughput sequencing. Black dots in the map indicate the WT amino acid and gray tiles indicate missing data. Position-averaged activity scores are shown in the bar graph above the heatmap. The average activity score at each position is shown in the bar graph above the heatmap. f, Scatterplot showing activity score correlations between the two independent replicates shown in e (Pearson’s R = 0.80). g, Individually assessed pErk levels measured by western blotting (n=3) compared to activity scores determined with the LABEL-seq activity assay for a panel of BRaf variants (Pearson’s R = 0.95). Error = SEM. h, The number of activating variants at each position in the untagged BRaf variant library (represented as spheres) projected onto the (left) inactive (PDB ID: 6NYB) and (right) active conformations (PDB ID: 4MNE) of BRaf’s kinase domain. The number of activating variants at a position is represented by the shade of red: white = 0 activating variants, brightest red = 19 activating variants. G466, S467, and F468 are not resolved in the active conformation of BRaf’s kinase domain. i, CRaf-promoted, BRaf variant activity at each position in the untagged BRaf variant library. CRaf-promoted activity scores were calculated by determining the difference between BRaf variant activity scores, which were each normalized to BRaf WT in the absence or presence of co-expressed CRaf. Positions highly conserved amongst protein kinases are shown in green. Gray dashed line indicates the WT score (0). Positive and negative values indicate more or less activity promoted by CRaf, respectively. j, Position-averaged CRaf-promoted activity scores projected onto the structure of the autoinhibited BRaf:Mek complex (PDB ID: 6NYB) bound to ATP-γ-S. Purple spheres are scores less than 0 (decreased activity with CRaf) and green spheres are scores greater than 0 (increased activity with CRaf).

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Activity Assay, Sequencing, Expressing, Variant Assay, Cell Culture, Lysis, Next-Generation Sequencing, Western Blot

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, General schematic of the proximity labeling-based LABEL-seq interaction assay. b, Biotinylation levels of Flag-tdMCP-BRaf I666R (left) and WT (right) in HEK293 cells co-expressing TurboID-Mek1 and labeled with biotin for 90 min. c , Biotinylation levels (represented as the ratio of streptavidin and Flag barcode sequence counts) of Flag-tdMCP-BRaf WT (blue circles), I666R (teal squares) and background control Flag-tdMCP-BTK (pink triangles) in HEK293T cells co-expressing TurboID-Mek1 and MS2-circRNAs, and treated with biotin for 0, 5, or 45 min. Following biotin treatment, cells were lysed, pooled, parallel streptavidin and Flag enrichments were performed, co-enriched barcodes were quantified by high-throughput sequencing, and the ratio of streptavidin and Flag counts for each co-enriched MS2-circRNA barcode sequence was calculated (n=3 barcodes/treatment, mean +/- SEM, *P<0.0332, **P<0.0021, ***P<0.0002, ****P<0.0001). d , Biotinylation levels of Flag-tdMCP-BRaf WT in HEK293T cells co-expressing TurboID-CRaf, treated with DMSO (left) or type 2 inhibitor LXH-254 (right) for 4 h, and then labeled with biotin for 90 min. e, Biotinylation levels (represented as the ratio of streptavidin and Flag barcode sequence counts) of Flag-tdMCP-BRaf WT and Flag-tdMCP-BTK in HEK293T cells co-expressing TurboID-CRaf and MS2-circRNAs, which were treated DMSO or GDC-0879 for 2 h followed by labeling with biotin for 0, 5, 15, or 45 min. Following biotin addition, cells were lysed, parallel streptavidin and Flag enrichments were performed, and the ratio of streptavidin and Flag counts for each co-enriched MS2-circRNA barcode sequence was calculated using high-throughput sequencing. f, Biotinylation levels (represented as the ratio of streptavidin and Flag barcode sequence counts) of Flag-tdMCP-BRaf WT and Flag-tdMCP-BTK in HEK293T cells co-expressing TurboID-BRaf and MS2-circRNAs, which were treated DMSO or GDC-0879 for 2 h followed by labeling biotin for 0, 5, or 45 min. Following biotin addition, cells were treated as described in e . For e , f (n=2-3 barcodes/treatment, mean +/- SEM, *P<0.0332, **P<0.0021, ***P<0.0002, ****P<0.0001) g , Schematic of the LABEL-seq assay for the multiplexed measurement of intracellular BRaf variant interactions. h, Sequence-CRaf interaction map for variants in the tdMCP-BRaf library co-expressed with TurboID-CRaf and labeled with biotin for 5 min. The interaction scores shown are the average of two replicates, each involving an independent cell culturing, biotin labeling, parallel streptavidin and Flag enrichments, and quantification with high-throughput sequencing. Black dots in the map indicate the WT amino acid and gray tiles indicate missing data. Position-averaged CRaf interaction scores are shown in the bar graph above the heatmap. i, The number of variants at each position that were classified as increased CRaf interaction in h projected onto a structure of BRaf’s kinase domain within the active BRaf dimer complex (PDB: 4MNE). White = 0 increased CRaf interaction variants, brightest red = 19 increased CRaf interaction variants. j, Sequence-Mek1 interaction map for variants in the tdMCP-BRaf library co-expressed with TurboID-Mek1 and labeled with biotin for 5 min. The Mek1 interaction scores shown are the average of two replicates, each involving an independent transfection, biotin labeling, parallel streptavidin and Flag enrichments, and quantification with high-throughput sequencing. Black dots indicate the WT amino acid and gray tiles indicate missing data. Position-averaged Mek1 interaction scores are shown in the bar graph above the heatmap. k, Position-averaged CRaf interaction scores for each position in the tdMCP-BRaf variant library (represented as spheres) projected onto a structure of autoinhibited BRaf bound to ATP-γ-S (PDB ID: 6NYB). The shade of red indicates the position-averaged CRaf interaction score: white = 1, brightest red > 3.2. i, Position-averaged Mek1 interaction scores projected onto the structure of the autoinhibited BRaf complex (PDB ID: 6NYB). The shade of blue indicates the position-averaged Mek1 interaction score: white = 1, darkest blue < 0.5.

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Labeling, Expressing, Sequencing, Control, Next-Generation Sequencing, Variant Assay, Cell Culture, Transfection

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, Biotinylation levels of Flag-tdMCP-BRaf variants in HEK293 cells co-expressing TurboID-Mek1 (top) or miniTurbo-Mek1 (bottom), treated with 10 μM cobimetinib or DMSO for 4 h, and labeled with biotin. b, Biotinylation levels of Flag-tdMCP-BRaf variants in HEK293 cells co-expressing TurboID-CRaf (top) or miniTurbo-CRaf (bottom), treated with 10 μM LXH254 or DMSO for 2 h, and labeled with biotin. ( c-e), Biotinylation levels (represented as the ratio of streptavidin and Flag barcode sequence counts) of Flag-tdMCP-BRaf variants and the background control Flag-tdMCP-BTK in HEK293T cells co-expressing TurboID-Mek1 (c), TurobID-CRaf (d), or TurboID-BRaf (e) and MS2-circRNAs. Cells were cultured in regular or dialzyed FBS and treated with biotin for 0, 5, or 45 min. Following biotin treatment, cells were lysed and pooled, parallel streptavidin and Flag enrichments were performed, co-enriched barcodes were sequenced by high-throughput sequencing, and the ratio of streptavidin and Flag counts for each co-enriched MS2-circRNA barcode sequence was calculated (n=3 barcodes/treatment, mean +/- SEM, *P<0.0332, **P<0.0021, ***P<0.0002, ****P<0.0001). Whole-cell lysate samples were reserved for western blots, are shown below the bar graph, and also correspond to , , . f, Biotinylation levels of Flag-tdMCP-BRaf variants in HEK293T cells co-expressing TurobID-CRaf and MS2-circRNAs, treated with a titration of GDC-0879 for 90 min then labeled with biotin. Labeled cells were lysed, samples corresponding to the same protein variant were pooled, parallel streptavidin and Flag enrichments were performed, and co-enriched barcodes were sequenced by high-throughput sequencing. For each protein variant, the background is subtracted from each ratio of barcode sequence counts derived from streptavidin and Flag enrichments then normalized to the signal at the maximum drug concentration (n=3 barcodes/concentration, error = SEM). Representative western blots are shown below.

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Expressing, Labeling, Sequencing, Control, Cell Culture, Next-Generation Sequencing, Western Blot, Titration, Variant Assay, Derivative Assay, Concentration Assay

Journal: bioRxiv

Article Title: Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

doi: 10.1101/2024.04.19.590094

Figure Lengend Snippet: a, Scatterplot showing CRaf interaction score correlations between 5 min and 45 min biotin labeling regimes (Pearson’s R = 0.86). Each score is the average of two replicates corresponding to parallel biotin labeling, streptavidin and flag enrichments, and quantification by high-throughput sequencing. b, Scatterplot showing CRaf interaction score correlations between the two 5 min biotin labeling replicates shown in (Pearson’s R = 0.96). c, CRaf interaction scores for previously characterized activating BRaf variants. Mean barcode score +/- SEM for each replicate. Assays were performed on separate days and replicates each involved independent biotin labeling, parallel streptavidin and Flag enrichments, and barcode sequence quantification by high-throughput sequencing. d, LABEL-seq CRaf interaction score distributions for all synonymous WT and nonsynonymous BRaf variants. The gray dashed line indicates the CRaf interaction score value (>2 standard deviations of the synonymous distribution higher and lower than WT) we defined as increased and decreased interaction, respectively. The black dashed line is the BTK score. e, CRaf interaction scores for BRaf variants classified as increased CRaf interaction (51.5%), Wild Type-like (43.4%), or decreased CRaf interaction (5.1%). f, Scatterplot comparing activity scores (with CRaf) to CRaf interaction scores. g, Scatterplot showing Mek1 interaction score correlations between the two independent replicates shown in (Pearson’s R = 0.80). h, Mek1 interaction scores for BRaf variants classified as increased Mek1 interaction (6.0%), Wild Type-Like (65.6%), or decreased Mek1 interaction (28.4%). i, LABEL-seq Mek1 interaction score distributions. The gray dashed line indicates the Mek1 interaction score value (>2 standard deviations of the synonymous distribution higher and lower than WT) we defined as increased and decreased interaction, respectively. The black dashed line is the BTK score. j, Mek1 interaction scores for the decreased interaction control variant I666R and Flag-tdMCP-BTK. Average barcode score +/- SEM. k, Co-immunoprecipitations of HA-Mek1 and Flag-BRaf variants (without tdMCP). LABEL-seq Mek1 interaction scores are shown above the plot (n=3, error = SEM). l, The number of variants at each position that were classified as decreased Mek1 interaction in projected onto a structure of the autoinhibited BRaf complex (PDB ID: 6NYB). m, LABEL-seq Mek1 interaction and activity (with CRaf) scores. Variants at select positions within the ATP-binding pocket are highlighted. Notably, these BRaf variants possessed a range of activity scores but similarly decreased Mek1 interaction scores, indicating that differences in Mek1 interactions cannot solely be attributed to the formation of pMek, which has a lower affinity for BRaf . Scores to the right of the vertical dashed line are classified as activating and scores below the horizontal dashed line are classified as decreased Mek1 interaction.

Article Snippet: To generate a library of barcodes, attB plasmid containing genes for tdMCP-protein fusions and a MS2-circRNA barcode were digested overnight at 37°C to remove existing barcode sequence (2 μg plasmid, 5 μL 10x CutSmart buffer, 2 μL BsrGI-HF (NEB cat#R3575S), nuclease-free water to 50uL).

Techniques: Labeling, Next-Generation Sequencing, Sequencing, Activity Assay, Control, Variant Assay, Binding Assay