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