Figure 1. Characterization of the sensitivity and specificity of class III ubiquitin conjugating enzyme (E2) antibodies. A: Dot blot assay using recombinant glutathione S-transferase (GST) or GST-E2 fusion proteins. The indicated recombinant proteins (10, 1, or 0.1 ng) were spotted on pieces of nitrocellulose paper in quintuplicate. The blots were blocked in 5% milk/TBST and then incubated with anti-GST, anti-UbcM3, anti-UBE2E2, anti-UbcM2, or a commercial antibody against human UbcM3 (anti-UbcH6). To the right of the blots is a diagram of the class III E2s highlighting the relative location of the conserved catalytic core domain (UBC), the number of residues in each protein, and the residue corresponding to the end of the unique N-terminal extension. B: Mouse retinal lysate (10 or 30 μg) was resolved by sodium dodecyl sulfate PAGE (SDS–PAGE) in quadruplicate, transferred to nitrocellulose, and probed with the indicated antibodies. The migration of UbcM2 and UbcM3 is indicated to the right of the anti-UbcH6 blot. Two distinct isoforms of UbcM3 are detected (arrows). The migration of molecular weight markers is indicated on the left. C: siRNA experiments in HeLa cells to demonstrate that targeted knockdown of UbcM2 results in loss of the band denoted as UbcM2 but does not affect UbcM3 expression (left blot), and targeted knockdown of UbcM3 results in loss of detection of both isoforms of the enzyme (right blot). The migration of molecular weight markers is indicated to the left of the blots.