Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

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Article Snippet: Rabbit monoclonal anti-p38 antibody Clone D13E1 , Cell Signaling Technology , Cat# 8690s; RRID:AB_10999090.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Reverse Transcription, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Gene Expression, CRISPR, Software, Sequencing, Modification

Journal: iScience

Article Title: Mitochondria-targeted cancer analysis using survival and expression: Prioritizing mitochondrial targets that alleviate pancreatic cancer cell phenotypes

doi: 10.1016/j.isci.2024.110880

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Article Snippet: Mouse monoclonal anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Recombinant, Modification, Ligation, Transfection, Infection, Electron Microscopy, Protease Inhibitor, Negative Control, shRNA, Sequencing, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Regulation of MYC by CARD14 in human epithelium is a determinant of epidermal homeostasis and disease

doi: 10.1016/j.celrep.2024.114589

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ERK (rabbit) (WB (1:1000)) , Cell Signaling Technologies, Danvers, MA , Cat# 4695; RRID: AB_390779.

Techniques: FLAG-tag, Plasmid Preparation, Recombinant, Modification, Transfection, Staining, Protease Inhibitor, Blocking Assay, Western Blot, Stripping, XF Assay, Reporter Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Sequencing, Cloning, Mutagenesis, Software, Membrane

Journal: eLife

Article Title: Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities

doi: 10.7554/eLife.78629

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Article Snippet: Antibody , Anti-pErk1/2-Thr202/Tyr204 (rabbit polyclonal) , Cell Signaling Technology , Cat# 4370; RRID: AB_2315112 , WB (1:500).

Techniques: Knock-Out, Transgenic Assay, Recombinant, Plasmid Preparation, Cloning, Sequencing, DC Protein Assay, Transformation Assay, Isolation, Reverse Transcription, Protease Inhibitor, Western Blot, Software

Journal: eLife

Article Title: Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes

doi: 10.7554/eLife.65512

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Article Snippet: Antibody , p44/42 MAPK (Erk1/2) (Total Erk) (Rabbit polyclonal) , Cell Signaling Technology , 9102 , WB (1:1000).

Techniques: Control, Recombinant, Flow Cytometry, Imaging, Plasmid Preparation, Software, Sequencing

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: A A schematic diagram of the Hog1 MAPK signaling pathway. B Analyses of Hog1 phosphorylation by immunoblotting with anti‐phospho‐p38 (Hog1‐P) and anti‐Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207, and KY594‐1. C Analyses of Hog1 phosphorylation by Phos‐tag band‐shift assay. Yeast strain KY594‐1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 (Hog1‐P [%]) were calculated as explained in Materials and Methods and are shown beneath the panel. D Analyses of Hog1 phosphorylation by immunoblotting with anti‐phospho‐p38 (Hog1‐P) and anti‐Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single‐copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild‐type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (−) 1 M NaCl for 5 min. E–H Analyses of Hog1 phosphorylation by Phos‐tag band‐shift assay. Yeast strains (E) KY603‐3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. I Comparison of the NaCl dose–responses of Hog1 activation by various strains. Phos‐tag band‐shift assays shown in (C and E–H) were independently repeated three times, and average values were plotted. Data information: (C and E–H) Representative results from three independent experiments. (I) Error bars are SEM ( n = 3). Source data are available online for this figure.

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Electrophoretic Mobility Shift Assay, Transformation Assay, Mutagenesis, Plasmid Preparation, Incubation, Comparison, Activation Assay

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: A Alignment of the amino acid sequences of the CD (green) and L16 (pink) domains of yeast Hog1 and mammalian p38α. The alpha helix αL16 forms the core of the L16 domain (Wang et al , ). B Schematic diagrams of Hog1‐WT and its deletion constructs used in this study. C Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416‐FLAG‐Hog1 (WT) or its indicated deletion derivatives. FLAG‐Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti‐phospho‐p38 (for Hog1‐P; upper panel) or anti‐FLAG (for total FLAG‐Hog1; lower panel) D–G Phos‐tag band‐shift assay of Hog1 phosphorylation. Yeast strain (D and E) KT235 or (F and G) KT290 carrying the single‐copy expression plasmid YCplac22I’‐Pbs2 S514D/T518D was transformed with either pRS416‐Hog1 (WT) or its indicated mutant derivatives and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarize the averages of three independent experiments. H Phos‐tag band‐shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single‐copy expression plasmid pRS416‐Hog1 (WT) or pRS416‐Hog1‐ΔL16 and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: (E, G, and H) Error bars are SEM ( n = 3). Source data are available online for this figure.

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Construct, Western Blot, Phospho-proteomics, Transformation Assay, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Expressing, Plasmid Preparation, Mutagenesis

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: KT299 ( MAT a hkr1 Δ msb2 Δ ssk2 / 22 Δ) was exposed to the indicated concentrations of α‐factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting. KT299 ( MAT a ssk2 / 22 Δ hkr1 Δ msb2 Δ) was exposed to the indicated concentrations of α‐factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average values of four independent experiments from (B) were plotted. HM06‐1 ( MAT a ΔS/O/H/M ssk2 / 22 Δ) was exposed to the indicated concentrations (log scale) of α‐factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average values of three or more independent experiments were plotted. KT306 ( MAT a hkr1 Δ msb2 Δ ssk2 / 22 Δ hog1 Δ) was transformed with pRS416‐Hog1 (WT) or pRS416‐Hog1‐N149H D162G (N/H D/G) and was exposed to 10 μM α‐factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average of three independent experiments is plotted. A schematic model showing that the lack of osmotic enhancement of the Pbs2‐Hog1 reaction prevents the pheromone‐to‐Hog1 crosstalk. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy‐p38 (Tomida et al , ) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in . Distribution of p38 activity in individual cells from sets (a)–(d) in (G). Data information: (B) Representative results from three independent experiments. (C–E) Error bars are SEM: (C) n = 4; (D), n = 3 or more; and (E) n = 3. (G) Scale bars: 20 μm. (H) Statistics, Student's two‐tailed t ‐test. Source data are available online for this figure.

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Electrophoretic Mobility Shift Assay, Transformation Assay, Activation Assay, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K

doi: 10.15252/embj.2019103444

Figure Lengend Snippet: Alignment of the amino acid sequences around the DFG motif of various MAP kinases. The positions of N149 and D162 in Hog1, the β7 and β8 strands, the DFG motif, the activation loop, and activating phosphorylation sites (the TXY motif) are indicated. The sequences of the mouse p38α and yeast Hog1 are highly conserved in this segment (23 residues out of 33 are identical, and the other residues show mostly conservative changes). Sc, Saccharomyces cerevisiae ; Mm, Mus musculus (mouse). The 3D structure of the mouse p38α MAPK (left), and an annotated enlargement of the relevant segment (residues 151–183; right). The corresponding amino acid sequence is shown in (A). Side chains of N155, D168, and activating phosphorylation sites T180 and Y182 are also shown. N155 and D168 correspond to, respectively, the yeast Hog1 residues N149 and D162, whose mutations created the constitutively enhanced phenotype. The coordinate data were from PDB (ID 5UOJ) (Wang et al , ) and were visualized using the MOLMOL program (Koradi et al , ). The 3D structure of the mouse p38α MAPK showing the spatial relationship between the L16 domain and the DGF motif. Four side views of the mouse p38α are shown, each of which was rotated 90° from the previous one around the vertical axis. Following segments are highlighted by coloring: the DFG motif (brown), the CD domain (green), and the L16 domain (pink).

Article Snippet: Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology).

Techniques: Activation Assay, Phospho-proteomics, Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Boxplots of growth phenotypes for PC9-Cas9-mCherry cells expressing the indicated pgRNA compared to PC9-Cas9-GFP cells expressing a double-safe-targeting control pgRNA. Boxes indicate mean ± SEM of six biological replicates, which are shown as overlaid points. Growth phenotype is defined as the log 2 -scaled ratio of mCherry:GFP cell counts at the late time point compared to the day 1 mCherry:GFP cell counts. Expected DKO phenotypes are the sum of single KO growth phenotypes. The expected and observed DKO phenotypes were compared using a one-tailed t test. Data shown are for the time point with the most extreme difference between expected and observed DKO growth phenotypes, termed the late time point: CCNL1/CCNL2 (day 12), CDK4/CDK6 (day 7), MEK1/MEK2 (day 11), and OXSR1/STK39 (day 10). Full time course data are shown in . (B) Fluorescence microscopy images of competitive fitness assays on early (day 1) and late time points as indicated above for (A). Scale bar, 100 μM. (C) Western blot validation of single KO and DKO pgRNA-induced gene inactivation. For CCNL1, pie charts of percent mutant alleles based on next-generation sequencing are shown due to lack of a suitable CCNL1 antibody for western blotting. Additional genomic DNA-level validation data are presented in . See also and and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Expressing, Control, One-tailed Test, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Mutagenesis, Next-Generation Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Rank plot of target-level GI scores in HeLa cells. Table insert, top synthetic lethal paralogs based on GI score. (B) Volcano plot of target-level GI scores in HeLa cells. FDR indicates the multiple hypothesis-adjusted p values from a two-tailed t test . Blue, synthetic lethal paralog GIs with GI < −0.5 and FDR < 0.1; red, buffering paralog GIs with GI > 0.25 and FDR < 0.1. (C) Scatterplot of target-level GI scores for paralog pairs in PC9 versus HeLa cells. Blue, synthetic lethal paralog pairs with GI < −0.5 and FDR < 0.1 in either PC9 or HeLa cells; gray, all paralog pairs with GI ≥ −0.5 or FDR ≥ 0.1. (D) CRISPR scores for representative synthetic lethal paralog pairs identified in the PC9 and HeLa cell screens. Top row: data shown are the mean CRISPR score for each single KO or DKO target across three biological replicates with replicate data shown in overlaid points. Shared synthetic lethal paralogs (e.g., CCNL1/CCNL2 and MEK1/MEK2 ) have FDR < 0.1 in both cell lines; PC9-specific paralogs (e.g., CDK4/CDK6 and OXSR1/STK39 ) have FDR < 0.1 in PC9 only; and HeLa-specific paralogs (e.g., GFTP1/GFPT2 and SOS1/SOS2 ) have FDR < 0.1 in HeLa only. Dashed lines indicate CRISPR score < −0.5. Bottom row: paralog gene expression in PC9 and HeLa cells from RNA-seq analysis. Dashed lines indicate log 2 (TPM) = 1, the threshold for gene expression. (E) Boxplots comparing the effect of CRISPR-mediated KO of the indicated gene in DepMap cell lines with high (top quartile) compared to low (bottom quartile) copy number of its paralogous gene. For boxplots, the middle line, hinges, notches, and whiskers indicate the median, 25th/75th percentiles, 95% confidence interval, and data points within 1.5× the interquartile range from the hinge, respectively. p values were computed using a two-tailed Wilcoxon rank-sum test. CRISPR score and copy number data were obtained from DepMap. (F) As in (E), but for gene expression. (G) Bar plot indicating the p values (computed using a two-tailed Wilcoxon rank-sum test) obtained by comparing the effect of a single paralog KO to the copy number (as in E) or gene expression (as in F) of its pair across human cancer cell lines profiled by DepMap. Bar color indicates whether each pair was synthetic lethal in PC9 only, HeLa only, or both cell lines in the pgPEN screens. Dashed line indicates p = 0.05. See also and , , and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Two Tailed Test, CRISPR, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

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Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Software