Journal: International Journal of Molecular Sciences

Article Title: E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

doi: 10.3390/ijms22115712

Figure Lengend Snippet: Analysis of SPL2cyt and MBP-SPL2cyt auto-ubiquitination activity in vitro. ( A ) Enzymatic assay of SPL2cyt in the absence or presence of La 3+ at indicated molar ratios. ( B ) Activity of zinc-free (apo) MBP-SPL2cyt after incubation with Zn or La ions. For comparison of enzymatic activity, holo MBP-SPL2cyt is also shown. ( C ) Effects of indicated mutations in the putative LBT region of MBP-SPL2cyt (D316R and D317R) and RING (C370A) on auto-ubiquitination activity. Wild-type MBP-SPL2cyt protein (WT) is shown for comparison. All Western blots were analyzed with anti-ubiquitin antibodies to detect free ubiquitin and polyubiquitination products. ( D ) CD spectra and structural changes in SPL2cyt upon removal of Zn 2+ and subsequent incubation with either Zn or La ions.

Article Snippet: Antibodies were as follows: anti-Human Ubiquitin antibody (clone 83406, Biotechne, Minneapolis, MN, USA), 1:5000 dilution in TBS supplemented with 1% non-fat milk; anti-Mouse IgG HRP-coupled secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:10,000 dilution in TBS supplemented with 0.5% non-fat milk.

Techniques: Activity Assay, In Vitro, Enzymatic Assay, Incubation, Comparison, Western Blot, Circular Dichroism

Journal: International Journal of Molecular Sciences

Article Title: E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

doi: 10.3390/ijms22115712

Figure Lengend Snippet: Selected sequences retrieved from BLAST searches of E3 ligases harboring LBT in plants (green), animals (black), and primates (red). ( A ) Lanthanide-binding motifs(cyan) present in some plant and animal E3 ligases. The ubiquitin-associated (UBA) domain of cIAP2 is highlighted in yellow. The caspase activation and recruitment domain (CARD) is shown as gray rectangles. ( B ) RING domains of selected E3 ligases. White letters on a red background represent strictly conserved residues; similar residues are shown as red letters on a white background in boxes.NCBI reference sequences are listed in Materials and Methods.

Article Snippet: Antibodies were as follows: anti-Human Ubiquitin antibody (clone 83406, Biotechne, Minneapolis, MN, USA), 1:5000 dilution in TBS supplemented with 1% non-fat milk; anti-Mouse IgG HRP-coupled secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:10,000 dilution in TBS supplemented with 0.5% non-fat milk.

Techniques: Binding Assay, Activation Assay