Figure 1
From: Characterization of MUDENG, a novel anti-apoptotic protein
MuD domain analysis and generation of a MuD monoclonal antibody (MAb)-recognizing domain 1 of MuD. (a) The hydrophilicity score was calculated based on the methods of Goldman, Engelman and Steitz (GES)25 and Kyte–Doolittle (KD)26 using the CLC Genomics Workbench program (http://www.clcbio.com/products/clc-genomics-workbench/), which was also used for calculating the isoelectric point (pI). The hydrophilicity of the MuD amino acid sequence (NCBI Reference Sequence: NM_018229.3) was evaluated using the GES (solid black line) and KD (dotted line) methods;25, 26 two hydrophilic regions (domain 1, pI=6.82; domain 3, pI=8.34) surround a non-hydrophilic region (domain 2, pI=4.32). (b) Domain 1 of MuD was expressed in Escherichia coli BL21 (DE3) using pET23dw-His-MuD and then purified using His-bind resin27 (Novagen, San Diego, CA, USA). The purified MuD protein was used to generate MAb; immunization, cell fusion and selection of hybridoma clones, and production and purification of the MAb were performed according to the standard procedures.28 A mouse MuD MAb (C22B3) produced from one of the hybridomas was used for the experiments. Green fluorescent protein (GFP)-tagged MuD deletion mutants (pEGFPC1-MuD AA 1-490; AA 41−490; AA 81−490; AA 121−490) were generated by PCR. All cDNA constructs were verified by DNA sequencing and expressed in HCT-116 cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) by transient transfection using Lipofectamine2000 (Invitrogen, Gaithersburg, MD, USA). Anti-GFP was purchased from Santa Cruz (Dallas, TX, USA) and horseradish peroxidase-conjugated goat anti-mouse IgGs were obtained from Jackson ImmunoResearch Laboratories (West Groove, PA, USA).
