Figure 1

BCA2 is co-expressed with and binds to both hHR23a and 14-3-3σ. [A] The bolded black amino acids represent key residues which are imperative to the structural integrity of the BZF and RING domains. The bolded yellow residues indicate amino acids which have been modified to alanine by site-directed mutagenesis. The BZF domain (blue) binds ubiquitin and is also the site of BCA2 auto-ubiquitination (ubiquitinated lysine residues are shown in red). The AKT domain (green) is site of AKT-mediated phosphorylation of BCA2, predicted by in silico analysis. Serine residues (yellow) are likely phosphorylated, and have been mutated to alanine in the BCA2 S132, 133A mutant. The RING domain (orange) is BCA2 autoubiquitination. The yellow cysteine residues were mutated to alanine to create ligase-dead BCA2 RING mutant. [B] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2, BZF (C22/25A), S132/133A, RING (C228/231A) or GST-empty vector along with myc/his-tagged hHR23A. The top panel shows the result of GST-pulldown for BCA2, probed for myc-hHR23a. Whole cell GST blots are shown at 2 different exposures due to the differences in expression between GST-tagged BCA2 variants or GST alone, where the expression is substantially higher, and thus required a lower exposure. The difference in hHR23a binding is not a result of hHR23a expression, as lanes show equal loading for hHR23a and tubulin. [C] BCA2 binds to GST-tagged 14-3-3σ from bacterial cell lysates. Purified recombinant wild-type BCA2 (lane 2) as well as the RING (lane 8) mutant (mt) bind to 14-3-3σ (even lanes) but not the GST- alone protein (odd lanes). [D] Immunoblots of lysates from HEK293T cells which were co-transfected with FLAG-tagged BCA2 along with Xpress-tagged 14-3-3σ. BCA2 was introduced at 1 μg, 2 μg or 3 μg of plasmid DNA, while 14-3-3σ vector concentration was kept constant. The top panel shows increasing concentration of BCA2, the middle panel indicates a progressive decrease in expression of 14-3-3σ. β-Actin levels indicate decreasing 14-3-3σ is not an artefact of mis-loading (bottom part). [E] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2 along with myc/his-tagged hHR23A. BCA2 was introduced to the system in an increasing concentration, 1 μg, 2 μg or 3 μg, while the concentration of hHR23a vector was kept constant. The top panel shows increasing concentration of BCA2, the middle panel does not show any decrease in the expression of hHR23a, indicating there is no degradation. β-Tubulin levels indicate that the blot is equally loaded (third panel).