Journal: Cell calcium

Article Title: Ca 2+ -dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

doi: 10.1016/j.ceca.2021.102457

Figure Lengend Snippet: Fig. 1. Interaction of S100A6 with cofilin-1 in NIH3T3 fibroblasts. (A) Pull-down assay with the use of protein lysate from NIH3T3 cells and S100A6 affinity resin (upper panel) or empty resin (lower panel). Lanes: 1-input, 2-unbound fraction, 3-last wash, 4-first wash with 0.5 M NaCl, 5-last wash with 0.5 M NaCl, 6-first wash with 1 M NaCl, 7- last wash with 1 M NaCl, 8- elution in buffer containing EGTA. Fractions were analyzed by SDS-PAGE (15% gel) fol lowed by immunoblotting developed with anti- cofilin-1 antibody. (B) Co-immunoprecipitation of S100A6 with cofilin-1 from NIH3T3 cell lysate. 30 μg of protein lysate was used directly for immunoblotting (input; lane 1 in both upper and lower panel) and 2.5 mg of protein lysate was incubated with (upper panel) or without (control, lower panel) anti-S100A6 monoclonal antibody and then with protein A/G agarose. In both panels, lane 2 shows unbound fraction, lane 3-last wash and lane 4-elution. Proteins were identified by immunoblotting using anti- cofilin-1 antibody. (C) Presence of S100A6- cofilin-1 complexes in NIH3T3 cells studied by PLA. Complexes of examined proteins are visualized in red; cell nuclei, stained with DAPI, are in blue. Scale bar is 20 μm.

Article Snippet: For co-immunoprecipitation assays 2.5 mg of protein lysate from NIH3T3 cells, obtained using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer’s instruction was incubated with protein A/G-Agarose (Santa Cruz Biotechnology) for 1 h at 4◦C, as described by Jurewicz et al. [31].

Techniques: Pull Down Assay, SDS Page, Western Blot, Immunoprecipitation, Incubation, Control, Staining

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: (A, B) Mouse aortic endothelial cells (MAoECs) were isolated from WT and Terf2iphomo-EC-specific knockout (EKO) mice. After transduction with Ad-p90RSK-WT (+) or Ad-GFP (−) as a control for 12 h, expression of indicated proteins was analyzed by Western blotting using specific antibodies (A). Graphs represent densitometry data from immunoblots for selected proteins (B). Data represent mean ± SD (n = 3). **p <0.01. All data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. (C) D-flow–induced p90RSK activation simultaneously induces EC senescence and activation via TERF2IP S205 phosphorylation. TERF2IP S205 phosphorylation induces nuclear export of the TERF2IP-TRF2 complex, leading to senescence induced by telomere shortening/dysfunction and EC activation (i.e., NF-κB activation) induced by TERF2IP-IkB kinase binding in the cytosol. Telomere dysfunction or dislocation of the TERF2IP-TRF2 complex from telomeres precipitates trans-repression of the distinct reciprocal gene set, including MKRN1, enhancing EC senescence and activation under d-flow conditions.

Article Snippet: TERF2 siRNA was purchased from Santa Cruz (sc-3805).

Techniques: Isolation, Knock-Out, Transduction, Control, Expressing, Western Blot, Activation Assay, Phospho-proteomics, Binding Assay