Journal: Nature Communications

Article Title: MELK inhibition disrupts actin cytoskeleton and broadly restricts human coronavirus infections

doi: 10.1038/s41467-026-72615-1

Figure Lengend Snippet: a Identification of cofilin-1 associated with MELK by IP-MS. HEK293T cells were transfected with EV or FLAG-MELK expression plasmid. After 48 h, cell lysates were incubated with anti-FLAG-M2 antibody-conjugated agarose for coimmunoprecipitation. Bound proteins were eluted with 3×FLAG peptide and identified by mass spectrometry. Source data are provided as a Source Data file. b Schematic diagram illustrating the relationship and functions between cofilin-1 and profilin-1. c Expression and purification of MELK, β-actin and actin-binding proteins (cofilin-1 and profilin-1) from E. coli . Images were representative of three independent experiments. d Schematic diagram of in vitro luminescent kinase assay. e In vitro luminescent kinase assay with recombinant MELK (100 ng) and increasing amounts of β-actin, cofilin-1 and profilin-1. Phosphorylation of the substrate was monitored through ADP produced during the kinase reaction. Source data are provided as a Source Data file. f Identification of phosphorylation sites on cofilin-1 modified by MELK. The spectra of the modified peptides were identified in samples containing both MELK and cofilin-1, but not in samples containing solely cofilin-1, showing modifications at g S3, h T70, and i S119. j Viral infection decreased the interaction between MELK and cofilin-1. Huh7 cells were infected with HCoV-OC43 or HCoV-229E for 24 h. The interaction between MELK and cofilin-1 was examined by coimmunoprecipitation. The gray values were quantified using ImageJ. Images were representative of three independent experiments. Source data are provided as a Source Data file. k In vitro kinase assay with recombinant MELK (100 ng) and cofilin-1 (1000 ng). The phosphorylation of cofilin-1 at S3 and T70 was determined by western blot using the indicated antibodies. We generated an antibody specifically detecting cofilin-1 phosphorylation at T70. Images were representative of three independent experiments. Source data are provided as a Source Data file. l Overexpression of MELK enhanced the phosphorylation level of cofilin-1 at S3 and T70. Huh7 cells were transfected with EV or FLAG-MELK expression plasmid. After 24 h, the phosphorylation of cofilin-1 at S3 and T70 was determined by western blot using the indicated antibodies. The gray values were quantified using ImageJ and normalized to cofilin-1. Images were representative of three independent experiments. Source data are provided as a Source Data file. m – o MELK inhibition in both activity and expression decreased the phosphorylation level of cofilin-1 at S3 and T70. The phosphorylation of cofilin-1 at S3 and T70 was detected in m OTSSP167-treated, n siMELK-transfected and o sgMELK-transduced cells with indicated antibodies. The gray values as shown in ( j ). Images were representative of three independent experiments. Source data are provided as a Source Data file. Data presented in e are mean ± SEM of n = 5 independent biological replicates. Statistical significance was determined using two-way ANOVA followed by two-sided Dunnett’s multiple comparisons test (versus the 0 ng control group for each protein). Exact p values for Cofilin-1 at all tested concentrations are indicated in the figure. β-actin and Profilin-1 showed no significant differences at all concentrations. For β-actin, the exact p values versus 0 ng control were 0.1760 (100 ng), 0.1985 (500 ng), >0.9999 (1000 ng), >0.9999 (1500 ng), and 0.8204 (2000 ng). For Profilin-1, the exact p values versus 0 ng control were 0.9958 (100 ng), 0.7716 (500 ng), 0.9998 (1000 ng), 0.9997 (1500 ng), and 0.9949 (2000 ng). See also Supplementary Fig. .

Article Snippet: Profilin-1 recombinant protein (catalog no. PL7211) was obtained from ECM Biosciences.

Techniques: Protein-Protein interactions, Transfection, Expressing, Plasmid Preparation, Incubation, Mass Spectrometry, Purification, Binding Assay, In Vitro, Kinase Assay, Recombinant, Phospho-proteomics, Produced, Modification, Infection, Western Blot, Generated, Over Expression, Inhibition, Activity Assay, Control

Journal: The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques

Article Title: Utility of Repeat Endpoint Quaking-Induced Conversion Testing in Creutzfeldt-Jakob Disease.

doi: 10.1017/cjn.2022.306

Figure Lengend Snippet: Figure 2: Neuropathologic findings typical of Creutzfeldt–Jakob disease. Samples from the left temporal neocortex reveal patchy spongi- form change with select vacuoles abutting cort- ical neurons (A, arrowheads) (hematoxylin and eosin, scale bar = 50 um). Immunoperoxidase preparations confirm the presence of fine punc- tate and larger granular collections of abnormal prion proteins (B) (anti-PrPSc, ECM Biosciences, Versailles, Kentucky, scale bar = 50 um).

Article Snippet: Immunoperoxidase preparations confirm the presence of fine punctate and larger granular collections of abnormal prion proteins (B) (anti-PrPSc, ECM Biosciences, Versailles, Kentucky, scale bar= 50 um).

Techniques:

Journal: The Journal of Cell Biology

Article Title: The Rac activator Tiam1 is required for α3β1-mediated laminin-5 deposition, cell spreading, and cell migration

doi: 10.1083/jcb.200509172

Figure Lengend Snippet: Adhesion and signaling of WT and Tiam1−/− keratinocytes. (A) Keratinocytes were seeded on an exogenous LN5 or LN1 matrix. After 30 min, the number of adherent cells was quantified in an enzymatic assay. Phase-contrast images were taken before cell lysis. The values in the histogram are means ± SD. Note that both WT and Tiam1−/− keratinocytes hardly adhere to and spread on LN1. Bar, 50 μm. (B) Growth factor–starved WT and Tiam1−/− keratinocytes were detached from the culture dish and reseeded on a LN5-coated surface in growth factor–free medium. A sample of the cells in suspension was lysed and the attached cells were lysed after 10, 30, 60, and 180 min. Lysates were immunoblotted for phosphoY397-FAK. β-Actin was used as a loading control. (C) Cells seeded as described in B were lysed after 5, 15, and 45 min. Lysates were immunoblotted for phospho-Src, -Stat3, and -ERK1/2. β-Actin was used as a loading control.

Article Snippet: All ECM proteins except LN5 were coated to culturing dishes overnight at 4°C at the following concentrations: 10 μg/ml FN (isolated from human plasma); 10 μg/ml LN1 (Becton Dickinson); 10 μg/ml VN (Sigma-Aldrich); 20 μg/ml Col I (Vitrogen/Nutacon); and 25 μg/ml Col IV (Becton Dickinson).

Techniques: Enzymatic Assay, Lysis, Suspension, Control