Journal: Cell reports

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

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet:

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: 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: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet: (A) WT and GSDMD knockout (GSDMD −/− ) iBMDMs were primed with LPS and stimulated with 10 μM nigericin over a time course of 45 min. Cellular lysates were examined by western blot for changes in phosphorylation of the ERK substrate motif PXSP. (B) Direct ERK targets as identified by Ünal et al. were probed for enriched molecular functions using WebGestalt. (C) A Venn diagram describing the overlap between RNA-seq differentially expressed genes (from ) and the RNA binding subset of ERK substrates. (D) A plot of the density of phosphorylation sites per kilodalton of protein mass in the human proteome, based on the PhosphoSitePlus database. TTP has a reported 1.35 phosphorylation sites per kDa. (E) A heatmap showing relative expression levels of TTP target genes in THP-1 monocytes after 0, 2, or 4 h of nigericin stimulation. (F) Western blots are representative of at least three independent experiments.

Article Snippet: Rabbit monoclonal anti-phospho-MAPK substrate (PXSP) , Cell Signaling Technology , Cat# 2325; RRID:AB_331820.

Techniques: Knock-Out, Western Blot, Phospho-proteomics, RNA Sequencing, RNA Binding Assay, Expressing

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

Figure Lengend Snippet: (A) WT and GSDMD knockout (GSDMD −/− ) iBMDMs were primed with LPS and stimulated with 10 μM nigericin over a time course of 45 min. Cellular lysates were examined by western blot for changes in electrophoretic mobility of TTP. (B) TTP knockout iBMDMs were generated on a WT and GSDMD −/− background using CRISPR-Cas9 technology. Individual clones were assessed for knockout via sequencing and western blot. Knockout clones were then pooled and assessed via western blot (top). TTP −/− cells were then reconstituted with empty vector (EV), WT TTP, TTP S220A, or TTP 5S to A (bottom). (C) TTP −/− iBMDMs were reconstituted with empty vector or myc-TTP. After a 45-min nigericin time course, myc-TTP was immunoprecipitated from whole cell lysate and assayed via western blot for phosphorylation of the ERK substrate motif PXSP. (D) A schematic of TTP outlining its functional domains and highlighting its two PXSP sites. Sequences surrounding the PXSP sites are given for mouse, human, and rat TTP, as well as drosophila Tis11 (a TTP analog). All numbering refers to the mouse protein. NES, nuclear export signal. (E) HEK293T cells were transiently transfected with ERK1, constitutively active MEK1, and WT or phosphosite mutant TTP. TTP was immunoprecipitated from whole cell lysate and the pulldown was assayed for phospho-PXSP motifs via western blot. (F) TTP −/− iBMDMs were reconstituted with WT and S220A myc-TTP. After a 45-min nigericin time course, myc-TTP was immunoprecipitated from whole cell lysate and assayed via western blot for phosphorylation of the ERK substrate motif PXSP. Western blots are representative of at least three independent experiments.

Article Snippet: Rabbit monoclonal anti-phospho-MAPK substrate (PXSP) , Cell Signaling Technology , Cat# 2325; RRID:AB_331820.

Techniques: Knock-Out, Western Blot, Generated, CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Immunoprecipitation, Phospho-proteomics, Functional Assay, Transfection, Mutagenesis

Journal: Cell reports

Article Title: Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP

doi: 10.1016/j.celrep.2025.115340

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Article Snippet: Rabbit monoclonal anti-phospho-MAPK substrate (PXSP) , Cell Signaling Technology , Cat# 2325; RRID:AB_331820.

Techniques: Recombinant, CyQUANT Assay, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Clinical and Translational Medicine

Article Title: Topical TYK2 inhibitor ameliorates psoriasis‐like dermatitis via the AKT‐SP1‐NGFR‐AP1 pathway in keratinocytes

doi: 10.1002/ctm2.70256

Figure Lengend Snippet: The tyrosine kinase 2 (TYK2) inhibitor reduced the generation of proinflammatory genes in keratinocytes (KCs) through AKT‐SP1‐NGFR‐AP1 pathway. (A) Western blot analysis of phosphorylation and total protein expression of JNK and c‐Jun in HaCaT cells given specified treatments for 48 h. (B) Western blot analysis of phosphorylation and total protein expression of MAPK pathway (including ERK, P38 and JNK) and c‐Jun in HaCaT cells given specified treatments for 48 h. (C, D) Quantitative polymerase chain reaction (qPCR) analysis on mRNA expression of several proinflammatory mediators (C) and NGFR (D) in HaCaT cells transfected with or without shNGFR (the second sequence of shNGFR) in combination with cytokines (IL‐17A, IL‐22 and TNF‐α) stimulation for 48 h ( n = 5–6). (E) Violin plots show the expression of Ngfr along with several proinflammatory genes in the Supraspinous KCs. (F, G) Western blot analysis on the expression of specified proteins in HaCaT cells after different treatments for 48 h. Stimu: stimulation with cytokines IL‐17A, IL‐22 and TNF‐α. (H) Schematic diagram of nerve growth factor receptor (NGFR) promoter reporter plasmid construction. (I) Luciferase activity in HaCaT cells either transfected the empty (EV) or respective promoter reporter plasmid of NGFR ( n = 3). (J) Luciferase activity in HaCaT cells transfected with or without SP1 siRNA in combination with the specified promoter reporter plasmid of NGFR ( n = 3). (K) SP1‐binding site in NGFR promoter predicted by JASPAR. (L) Relative enrichment fold of NGFR promoter chromatin immunoprecipitated with SP1 in HaCaT treated with or without BMS‐986165 for 48 h analysed by qPCR ( n = 3). (M) Western blot analysis of the phosphorylation and total protein expression of AKT and SP1 in HaCaT cells after treated with BMS‐986165 for 48 h. (N) The diagram of regulatory mechanism of topical TYK2 inhibitor in psoriasis mouse model. ns, not significant. * p < .05, ** p < .01, *** p < .001, **** p < .0001 by one‐way analysis of variance (ANOVA) (C, D, I, J, L). Data are shown as mean ± standard error of the mean (SEM).

Article Snippet: Anti‐TYK2 (cat. ab223733, Abcam, 1:100), Phospho‐TYK2 (cat. PA5‐105556, Thermo Fisher, 1:1000), glyceraldehyde‐3‐phosphate dehydrogenase (cat. 60004‐I‐Ig, Proteintech, 1:3000), STAT3 (cat. 9139S, CST, 1:1000), Phospho‐STAT3 (cat. 9145S, CST, 1:1000), NGFR (cat. ab52987, Abcam, 1:1000), SAPK/JNK (cat. 9252S, CST, 1:1000), Phospho‐SAPK/JNK (cat. 4668S, CST, 1:1000), c‐Jun (cat. ab40766, Abcam, 1:1000), Phospho‐c‐Jun (cat. 3270S, CST, 1:1000), p44/42 MAPK (Erk1/2; cat. 4695S, CST, 1:1000), Phospho‐p44/42 MAPK (Erk1/2; cat. 4370S, CST, 1:2000), p38 MAPK (cat. 8690S, CST, 1:1000), Phospho‐p38 MAPK (cat. 4511S, CST, 1:1000), Akt (cat. 4691S, CST, 1:1000), Phospho‐Akt (cat. 4056S, CST, 1:1000), SP1 (cat. 9389S, CST, 1:1000) and Phospho‐SP1(cat. ab59257, Abcam, 1:1000) were used as primary antibodies.

Techniques: Western Blot, Phospho-proteomics, Expressing, Real-time Polymerase Chain Reaction, Transfection, Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay, Immunoprecipitation

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: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p < 0.05 compared to the negative control group.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Inhibition, Western Blot, Phospho-proteomics, In Vitro, Migration, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon up-regulates p38β expression in hepatic cells. Pokemon was delivered by expression plasmid pcDNA3.1(−)-Pokemon with pcDNA3.1(−) as a negative control. Pokemon silencing was triggered by si-RNA. ( a ) Targeted expression or silencing of Pokemon in HepG2 cells. Cells were collected at 60 h after transfection or silencing (Left panel). And the quantification of western blot data was displayed on the Right panel; ( b ) Real-time quantitative polymerase chain reaction (qPCR) at 48 h after transfection in HepG2 cells. Upper panel: Ectopic expression of Pokemon; lower panel: Silencing of Pokemon; ( c ) Ectopic expression of Pokemon in HL7702 cells. Upper panel: Western blot in which cells were lysed and total proteins were collected at 36, 48, 60 and 72 h, respectively. Lower panel: Real-time qPCR at 48 and 60 h after transfection; ( d ) Silencing of Pokemon in BEL7404 cells. Upper panel: Western blot; lower panel: Real-time qPCR. * p < 0.05 compared to the negative control. N.C. means negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon stimulates p38β promoter activity. ( a ) ChIP assays in HepG2 and BEL7404 cells. Protein-DNA complexes are immunoprecipitated either with anti-Pokemon antibody or anti-IgG as negative control, followed by PCR with primers specific to p38β promoter sequence and agarose-gel electrophoresis for visualization. Total lysates were used as the input samples and positive control; ( b ) Dual luciferase reporter assay. Luciferase activities were normalized to Renilla activity. Y axis stands for the relative fold changes of activity as the pcDNA3.1(−)-Pokemon plasmid increases. * p < 0.05 compared to the negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Activity Assay, Immunoprecipitation, Negative Control, Sequencing, Agarose Gel Electrophoresis, Positive Control, Luciferase, Reporter Assay, Plasmid Preparation

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

Figure Lengend Snippet:

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

Journal: Developmental cell

Article Title: Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1 P29S Melanoma Cells

doi: 10.1016/j.devcel.2019.04.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-MEK 1/2 , Cell Signaling , Cat#9122; RRID: AB_823567.

Techniques: Recombinant, Mutagenesis, Blocking Assay, Plasmid Preparation, Immunodetection, Staining, Imaging, Flow Cytometry, CRISPR, Sequencing, Knock-Out, Software, Immunofluorescence