Journal: Nature Communications

Article Title: Structures of complete extracellular assemblies of type I and type II Oncostatin M receptor complexes

doi: 10.1038/s41467-024-54124-1

Figure Lengend Snippet: a STAT3-luciferase reporter assay was performed in mouse NIH3T3 cells after treating the cells with short form mOSM WT and five mutant proteins with mutations at Site 3. The data shown are representative of 3 independent experiments with similar results. Error bars represent standard deviations. RLU: relative luminescence units. b WT hOSMR or six hOSMR mutants were stably overexpressed in human Ramos.2G6.4C10.stat3luc cells; STAT3-luciferase reporter assay was performed after treating the cells with E.coli -derived short form hOSM protein (R&D, #295-OM). The data shown are representative of 3 independent experiments with similar results. Error bars represent standard deviations. Source data are provided as a Source Data file.

Article Snippet: Human IMR32.stat3luc cells and mouse NIH3T3.stat3luc cells were plated overnight in assay buffer containing OptiMEM (Thermo Fisher, #51985), 1% Bovine Serum Albumin (Sigma, #A7030), and 0.1% Fetal Bovine Serum (FBS) (Seradigm, #97068-085), in white PDL-coated 384 well or 96 well plates (Corning, #354660 or #354651).The next day, cells were treated with indicated cytokines titrated in 1:3 dilution series for 6 hrs.

Techniques: Luciferase, Reporter Assay, Mutagenesis, Stable Transfection, Derivative Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Conformational plasticity links structural instability of NAA10 F128I and NAA10 F128L mutants to their catalytic deregulation

doi: 10.1016/j.csbj.2024.11.014

Figure Lengend Snippet: NAA10 F128L and NAA10 F128I are stably expressed, but show weak interaction with NAA15. (A) IMR32 cells were untransfected or transfected with clones of FLAG-tagged NAA10, NAA10 F128L , and NAA10 F128I for 24 h. Representative confocal immunofluorescence microscopy images showing expression of endogenous NAA10, FLAG-NAA10, FLAG-NAA10 F128L , and FLAG-NAA10 F128I . A significant number of IMR32 cells aggregated FLAG-NAA10 F128L . (B) Quantification of cells showing aggregated expression of endogenous NAA10, FLAG-NAA10, FLAG-NAA10 F128L , and FLAG-NAA10 F128I [100 cells/group in each of three independent experiments]. (C) IMR32 cells were transfected with FLAG-NAA10, FLAG-NAA10 F128L , and FLAG-NAA10 F128I for 24 h. Representative confocal immunofluorescence microscopy images of colocalization of FLAG-NAA10, FLAG-NAA10 F128L and FLAG-NAA10 F128I with endogenous NAA15. (D) Quantification of Pearson’s correlation coefficient of colocalization of FLAG-NAA10, FLAG-NAA10F128L, and FLAG-NAA10F128I with endogenous NAA15 as represented in 5 C [100 cells/group]. (E) IMR32 cells were transfected with FLAG-NAA10, FLAG-NAA10 F128L and FLAG-NAA10 F128I for 24 h. Representative immunoblots of FLAG-NAA10, FLAG-NAA10 F128L , FLAG-NAA10 F128I , and NAA15 from cell lysates with non-denaturing immunoprecipitation of FLAG-NAA10, FLAG-NAA10 F128L and FLAG-NAA10 F128I . (F) Relative densitometric quantification of the immunoblot bands of NAA15 with respect to immunoblot bands of FLAG-NAA10, FLAG-NAA10 F128L , and FLAG-NAA10 F128I in the immuno-enriched fractions of FLAG-NAA10, FLAG-NAA10 F128L , and FLAG-NAA10 F128I . Quantifications are shown as mean ± S.D.; P values are indicated. Scale bar in confocal microscopy images: 5 µm. Microscopy and immunoblot data are representative of at least three independent biological and technical experiments. Each data point of the acetylation assay is the average value of three technical replicates.

Article Snippet: Briefly, total mRNA was isolated from IMR32 cells using RNA extraction kit [ThermoFischer Scientific (TFS), 12183018 A], followed by the generation of cDNA pool from the mRNAs using oligo-dT (Eurofin genomics) primer and reverse transcription.

Techniques: Stable Transfection, Transfection, Clone Assay, Immunofluorescence, Microscopy, Expressing, Western Blot, Immunoprecipitation, Confocal Microscopy, Acetylation Assay