Journal: Nature Communications

Article Title: NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart

doi: 10.1038/s41467-022-33658-2

Figure Lengend Snippet: Immunoprecipitation (IP) was performed by using custom ELC antibodies covalently coupled to magnetic beads. Liquid chromatography followed by mass spectrometry (LC-MS) was used for identification. A Validation of ELC precipitation by immunoblot (IB) before performing LC-MS (see B and C ). B Classification of ELC enriched proteins identified by LC-MS. From 3452 spectra 110 proteins were identified in total. 106 proteins were found to interact with ELC. 3 different kinases were found. (Peptide FDR: 5%, minimum of two identified peptides per protein) C Ranking of ELC enriched proteins by foldchange of unique peptide counts enriched by ELC compared to unique peptide counts of the same protein captured by the isotype control (IgG Ctrl). Data of the screening experiment are shown without statistical analysis. Candidates were validated using independent methods. The string intersection score was calculated for known ELC interacting proteins . (NA: not available) D Two representative mass spectrometry spectra of unique peptides accepted to identify NIMA related kinase 9 (NEK9) (left panel) or calcium/calmodulin-dependent protein kinase type II subunit gamma (CamK2G) (right panel). The protein coverage is shown above (red boxes). E Validation of ELC-kinase interaction. Human myc-tagged ELC protein was overexpressed in human cells followed by specific immunoprecipitation (IP) and immunoblot (IB) analysis. Ca 2+ -influx was obtained by ionophore stimulation. One representative experiment is shown (see also Fig. ). F Schematic illustration of ascorbate peroxidase (APEX) catalyzed proximity labeling. Overexpression of ELC fused to APEX enables biotin-labeling of proteins in close proximity. The reaction is catalyzed by hydrogen peroxide (H 2 O 2 ). Streptavidin precipitation followed by LC-MS analysis identified NEK9 and CamK2G. The foldchange of unique peptide counts of biotin-labeled proteins in the proximity of ELC (APEX-ELC) compared to unique peptide counts of the same protein in the APEX-Ctrl sample is shown. Data of the validation experiment are shown without statistical analysis.

Article Snippet: For ELC-specific co-immunoprecipitation the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D) in combination with Dynabeads TM M-270 Epoxy (Invitrogen, #14302D) was used.

Techniques: Immunoprecipitation, Magnetic Beads, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Labeling, Over Expression

Journal: Nature Communications

Article Title: NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart

doi: 10.1038/s41467-022-33658-2

Figure Lengend Snippet: A NEK9 and CamK2G RNA expression in human total RNA tissue panel. Ribosomal protein large subunit P0 (RPLP0) was used as housekeeping gene. B NEK9 (left panel) and CamK2G (right panel) protein expression in piscine and human heart tissue to detect cross-species applicability. C mRNA expression profile of NEK9 and CamK2G in different human tissues from 4 weeks post-conception to adulthood . D , E Relationship of NEK9-ELC binding efficiency on intracellular Ca 2+ -concentration. NEK9-ELC interaction was analyzed after overexpression of myc-tagged ELC protein in human cells followed by specific immunoprecipitation (IP) and immunoblot (IB) analysis ( D ). Ca 2+ -influx was obtained by ionophore stimulation. Quantification of NEK9-ELC interaction dependent on Ca 2+ -influx illustrated as the averaged intensity of precipitated NEK9 protein normalized to actin protein expression of input protein lysate ( E ). Data are mean ± SD ( n = 3). ** P < 0.01 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test.

Article Snippet: For ELC-specific co-immunoprecipitation the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D) in combination with Dynabeads TM M-270 Epoxy (Invitrogen, #14302D) was used.

Techniques: RNA Expression, Expressing, Binding Assay, Concentration Assay, Over Expression, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart

doi: 10.1038/s41467-022-33658-2

Figure Lengend Snippet: Two heterozygous transgenic zebrafish lines were generated by CRISPR/Cas9. A NEK9 78del protein is lacking the ATP-binding domain shown in the 3D structure of NEK9 ( A ). NEK9 500del is harboring a frame shift mutation leading to a premature stop codon. Sanger sequencing results of the affected allele are shown in Supplementary Fig. . B , C Piscine ELC and mutated piscine myc-tagged NEK9 protein either lacking the ATP-binding domain (NEK9 78del ) or harboring a premature stop codon (NEK9 500del ) were overexpressed in HEK293 cells. The ELC-NEK9 interaction was analyzed by specific immunoprecipitation (IP) and immunoblot (IB) ( B ). Quantification of ELC-NEK9 interaction illustrated as averaged intensity of precipitated ELC protein normalized to ELC expression of input protein lysate ( C ). Data are mean ± SD ( n = 3). * P < 0.05; *** P < 0.001 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test. D , E Validation of ELC-NEK9 interaction. Human myc-tagged ELC protein and different deletion variants of human flag-tagged NEK9 protein were overexpressed and interaction intensity was quantified after IP and IB. Data are mean ± SD ( n = 3). ** P < 0.01 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test.

Article Snippet: For ELC-specific co-immunoprecipitation the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D) in combination with Dynabeads TM M-270 Epoxy (Invitrogen, #14302D) was used.

Techniques: Transgenic Assay, Generated, CRISPR, Binding Assay, Mutagenesis, Sequencing, Immunoprecipitation, Western Blot, Expressing