free software bioedit sequence alignment editor version 7 0 5 2  (Bioedit Company)

Journal: FEBS open bio

Article Title: CoCl 2 -triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299.

doi: 10.1002/2211-5463.13715

Figure Lengend Snippet: Fig. 4. The SIRT4 mutants K78R and K299R show a decreased polyubiquitination upon CoCl2-induced pseudohypoxia. (A) HEK293 cell lines expressing SIRT4-eGFP or the indicated SIRT4-eGFP mutants thereof were either untreated or subjected to CoCl2-induced pseudohypoxia for 24 h. SIRT4 variants were immunoprecipitated using anti-eGFP nanobody beads and further analyzed for the degree of polyubiquitination (poly-ub) using anti-Ubiquitin immunoblotting (upper panel). Immunoprecipitated SIRT4-eGFP proteins were detected on the same membrane using anti-eGFP antibodies (lower panel). (B) Relative quantification of polyubiquitination of SIRT4-eGFP mutants compared to wild-type SIRT4-eGFP using IMAGEJ-based densitometric evaluation. To determine statistical significance, a Two-Way ANOVA test followed by Tukey’s test was employed (n = 4; mean SD; *P < 0.05; ***P < 0.001). (C) SIRT4-specific conservation of lysines K78 and K299 (marked in red) within the human Sirtuin protein family. (D) Analysis of evolutionary conservation of K78 and K299 (marked in red) in SIRT4 homologs of ver- tebrates. Sequences in (C) and (D) were obtained from the UniProt database (www.uniprot.org). Sequence analysis was performed using the CLUSTALW multiple alignment method followed by the sequence alignment editor software BIOEDIT 7.2.5.

Article Snippet: Sequence analysis was performed using the CLUSTALW multiple alignment method followed by the sequence alignment editor software BIOEDIT 7.2.5.

Techniques: Expressing, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Membrane, Sequencing, Software