Journal: Biochimica et Biophysica Acta

Article Title: Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function

doi: 10.1016/j.bbadis.2014.03.006

Figure Lengend Snippet: a. Effects of wild-type and PDB mutant SQSTM1 over-expression on basal NF-κB activity. HEK293T cells were co-transfected with His-FLAG-SQSTM1 pcDNA3.1 constructs as indicated or empty vector (ev) pcDNA3.1 control along with NF-κB Firefly luciferase reporter and control Renilla luciferase construct . Cells were harvested 30 h after transfection and luciferase activity was measured. Firefly luciferase values were normalised to Renilla values. Experiments were performed in quadruplicate and assays were repeated on 4 separate occasions. Data is displayed as mean ± SD with p values indicating significance of difference compared with wild-type as indicated. The A427D mutant gave had the highest level of activation of all PDB mutants tested to date. b. Western blotting analysis (anti-SQSTM1, anti-β-actin) showed comparable expression levels of the different His-FLAG-SQSTM1 constructs (HF-SQSTM1) from a representative set of samples from a single NF-κB reporter assay.

Article Snippet: 24 h after the cells were seeded they were co-transfected with 0.2 ng/well of control Renilla luciferase plasmid (pRL0CMV; Promega), 100 ng/well of NF-κB Firefly luciferase reporter plasmid and 700 ng/well of His-FLAG-SQSTM1 construct (either wild-type or PDB-mutant).

Techniques: Mutagenesis, Over Expression, Activity Assay, Transfection, Construct, Plasmid Preparation, Control, Luciferase, Activation Assay, Western Blot, Expressing, Reporter Assay