Identification and characterization of two Nek4 isoforms. (A) cDNAs to Nek4 isoform 1 (Nek4.1) and Nek4 isoform 2 (Nek4.2) were amplified from HEK293T cells extracts. (B) cDNA to Nek4.2 encodes a protein of 781 residues which is very similar to Nek4.1 except for the regulatory domain where Nek4.1 shows a 46 amino acid residues long insertion, which is encoded by an Alu DNA sequence. (C) HEK293 Flp-In T-REx stable cell lines expressing cDNA for both isoforms of Nek4 fused to a Flag tag were lysed and an immunoprecipitation for the Flag tag was performed. The eluate was used for mass spectrometry and western blotting analyses. Silver staining to Nek4 isoforms and immunoprecipitated proteins (below). Western blot to identify Nek4 isoforms (upper panel). (D) Venn diagram showing exclusive or common interactors of both Nek4 isoforms. Bp: DNA ladder. MW: Protein ladder. KD: kinase domain (red). ALU: translated region from retrotransposonAlu. Flag φ: empty vector – negative control.