Journal: eLife

Article Title: Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel

doi: 10.7554/eLife.78301

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , D4476 , TargetMol , Cat#T2449; CAS: 301836-43-1 , CK1 inhibitor.

Techniques: Software

Journal: Chemistry & biology

Article Title: Inhibition of Wnt/β-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase Iδ/ɛ.

doi: 10.1016/j.chembiol.2011.01.015

Figure Lengend Snippet: Figure 4. Inhibition of CKId/3 Inhibits rmWnt-3a-Induced Phosphorylation of Human Dishevelled 2 and Blocks Signaling Through the Wnt/ b-Catenin Pathway (A) U2OS-EFC cells were treated with increasing concentrations of the CKI inhibitors IC261 (upper panel) and D4476 (lower panel) in the presence of 10 nM rmWnt-3a for 1 hr. Cell lysates were immunoblotted for human Dishevelled-2 (hDvl2) and b-actin. As a control for Wnt/b-catenin pathway inhibition, cells were cotreated with 10 nM rmWnt-3a and 1 mg/ml human Wnt inhibitory factor-1 (WIF1). See also Figure S3. (B) U2OS-EFC cells were treated with increasing concentrations of IC261 and D4476 in the presence of 10 nM rmWnt-3a for 3 hr, followed by measurement of b-galactosidase activity. Data are presented as mean ± SEM. See also Figures S4 and S5.

Article Snippet: D4476 and SB239063 were obtained from Tocris Cookson Inc. (St. Louis, MO).

Techniques: Inhibition, Phospho-proteomics, Control, Activity Assay

Journal: Nature Communications

Article Title: NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

doi: 10.1038/s41467-021-26142-w

Figure Lengend Snippet: a BMDMs transfected with the indicated siRNAs were treated with LPS followed by nigericin. Cell death was monitored by PI incorporation over time (for 140 min following nigericin treatment) and quantified by high content microscopy. Means of area under the curve normalized to non-targeting siRNA control and 1 SD are represented. b IL-1β and TNF secretions were measured by ELISA. c , d BMDMs transfected with Csnk1a1 siRNA were treated with LPS (6 h) followed by nigericin. IL-18 and TNF secretions were measured by ELISA ( c ). ASC specks were visualized and counted by immunofluorescence ( d ). Arrow, ASC specks. Scale bar, 20 μm. Quantification of >10 cells per replicates is shown (total cells n = 571 NT, 158 Csnk1a1 ). Means and 1 SD are represented. e GST-NLRP3 was incubated with GST-CSNK1A1, GST-CSNK2A1/GST-CSNK2B, or 6-His-GST-CAMK2B. In vitro phosphorylation was revealed by SDS-PAGE and autoradiography. Coomassie stainings serve as controls. f NLRP3 was ectopically expressed with HA-CSNK1A1 in 293T. HA-CSNK1A1 immunoprecipitates were analyzed for NLRP3. g BMDMs were treated with LPS (6 h). CSNK1A1 immunoprecipitates were analyzed for NLRP3. Lysate of Nlrp3 +/+ BMDMs incubated with isotype control and A/G-beads ( Nlrp3 +/+ +IgG) and lysates of Nlrp3 −/− BMDMs were used as negative controls. h NLRP3 WT or S806A mutant were ectopically expressed with HA-CSNK1A1 in 293 T. NLRP3 immunoprecipitates were analyzed for phospho-Ser by WB. i NLRP3-deficient U937 cells reconstituted with NLRP3 were treated with PMA and doxycycline for 16 h followed by D4476 and LPS (4 h). NLRP3 immunoprecipitates were analyzed for phospho-Ser by WB. Data are biological quadruplicates representative of two independent experiment ( a , c ), biological triplicates representative of four independent experiment ( b ), 11 technical replicates representative of two independent experiments ( d ), one representative of two ( e , h , i ) and three ( f , g ) independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple comparisons ( a ), ordinary two-way ANOVA with Tukey’s multiple comparisons ( c , d ) to corresponding non-targeting siRNA control; * p -value < 0.05; ** p -value < 0.01; *** p -value <0.001; **** p -value < 0.0001. Molecular weights are indicated in kDa ( e – i ). AUC area under the curve, NT non-targeting siRNA, Lys lysates, IP immunoprecipitates.

Article Snippet: The following reagents were used: MG132 (Sigma-Aldrich), E-64d (Enzo Life Science), VX765 (Invivogen), G5 (Merck), doxycycline (Sigma-Aldrich), LPS (O111:B4, Sigma-Aldrich), Pam3CSK4 (Invivogen), nigericin (Invivogen), ATP (Sigma-Aldrich), H-Leu-Leu-OMe (LLOMe, Santa Cruz Biotechnology), MSU (Invivogen), silica (U.S.Silica), poly(dA:dT) (Invivogen), flagellin (FLA-ST Invivogen), oridonin (Euromedex), and D4476 (Sigma-Aldrich), SP600125 (Selleck Chemical).

Techniques: Transfection, Microscopy, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Incubation, In Vitro, Phospho-proteomics, SDS Page, Autoradiography, Mutagenesis

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: Early phosphorylation of p53 in S20 induced by Resv is necessary for p53-stability in MCF-7 R cells. ( A ) MCF-7 R cells were treated with CDDP (6 μM) with or without Resv (100 μM) and ( B ) MCF-7 cells were treated with Resv (100 μM); both cell cultures were treated for 6 h with specific p53-pS20 site kinase inhibitors: CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM). Total and phospho-p53 contents are assessed by Western blot using antibodies directed against total p53 (DO-1) or against the specific phosphorylated residue on S20, as indicated. ( C ) MCF-7 and MCF-7 R cells were treated with a DMSO–ethanol vehicle as control or CDDP (6 μM) with resveratrol (100 μM) and cultured in combination with CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM) inhibitors for 48 h and were double-stained with Annexin V and propidium iodide (PI) followed by flow cytometry analysis to determine apoptotic cells. The viable cells are located in the lower left quadrant (double negative with Annexin V–/PI–). Apoptotic cells (Annexin V+/PI–) appear in the lower right (early apoptosis) and upper right (late apoptosis) quadrant of data plots. Data are presented as a percentage of the cell population. ( D ) The combined results of three independent cytometry analyses depicting the mean levels of total apoptotic cells are shown. Results are presented as the means ± SD. *** p < 0.001 by one-way ANOVA followed by Turkey’s Multiple Comparison test.

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Residue, Control, Cell Culture, Staining, Flow Cytometry, Cytometry, Comparison

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: In the MCF-7 resistant cell variant (MCF-7 R ), Resv attenuates phosphorylation in S15 and S46 of p53 by dephosphorylation and deactivation of ATM. However, it activates kinases CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20 (which is required to activate p53 in order to upregulate BAX and PUMA genes) and modifies the ratio between BCL-2/BAX expression. The BAX protein was increased while BCL-2 protein was decreased, restoring apoptosis and overcoming chemoresistance. On the other hand, the overexpression of BCL-2 in MCF-7 R cells after CDDP treatment maintains the chemoresistance and blocks apoptosis despite the phosphorylation of p53 in S15 and S46 and the upregulation of NOXA and PUMA .

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Variant Assay, Phospho-proteomics, De-Phosphorylation Assay, Expressing, Over Expression

Journal: PLoS Genetics

Article Title: Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds

doi: 10.1371/journal.pgen.1007850

Figure Lengend Snippet: (A) Lysates from NIH/3T3 stable cell lines expressing the dog, Myc-tagged wild-type (Wt) or mutant variant (Mut) DVL2, which is 23 aa shorter than wild-type exogenous DVL2, were analyzed by western blotting using an anti-c-Myc antibody. To assess the ability of the wild-type and mutant proteins to respond to WNT stimulation, cells were treated with WNT5A or WNT3A for 6 hours. Both treatments resulted in increased gel mobility shifts of the wild-type DVL2 protein, indicative of increased phosphorylation; this effect was reduced on the mutant DVL2 protein. (B) To confirm that the DVL2 gel mobility shifts observed in (A) were due to phosphorylation, cell lysates were subjected to mock treatment (30 min incubation at 37 C), or calf intestinal phosphatase (CIP) treatment (30 min incubation at 37 C in the presence of CIP) before separation by SDS-PAGE. The DVL2 gel mobility shifts above wild-type and mutant proteins were lost after CIP treatment, confirming that they are caused by phosphorylation. (C) To test whether the DVL2 gel mobility shifts observed in (A) were driven by casein kinase 1 (CK1), cells were treated with D4476, a CK1 inhibitor, for 1 hour prior to and concurrently during the Wnt stimulation for 6 hours. The DVL2 gel mobility shifts were lost after D4476 treatment, further indicating that they are caused by CK1-dependent phosphorylation. α-tubulin was used for loading controls. Cell lysates were normalized by BCA assays for total protein.

Article Snippet: For casein kinase 1 inhibitor treatment, cells were pre-treated with D4476 (APEXBIO catalog #A3342, 100nM final concentration) for 1h prior to WNT5A or WNT3A treatment.

Techniques: Stable Transfection, Expressing, Mutagenesis, Variant Assay, Western Blot, Phospho-proteomics, Incubation, SDS Page