FIGURE 2.

Expression of the RHBDD2 protein in transfected HEK293 cells and in mouse embryonic eyes, postnatal retina, and brain. A, schematic illustration of the pcDNA4/HisMax RHBDD2 expression construct. The vector contains the SP163 translational enhancer directly upstream of the ATG initiation codon, and the enterokinase (EK) cleavage site preceding the Rhbdd2 cDNA. B, Western blot of a crude protein extract obtained from cultured Rhbdd2-transfected cells using 7Rc (left lane) and the Xpress antibody (right lane). C, immunocytochemical detection of the RHBDD2 protein (green) after transient transfection of the expression vector into HEK293 cells. Propidium iodide was used to visualize the nucleus of the cells (red). Scale bar, 15 μm. D, retinal and brain protein extracts (60 μg each) from P21 and P90 mice were subjected to 8% SDS-PAGE under reducing conditions followed by immunoblotting using 7Rc. The lower panel shows 39-kDa protein bands in both tissue samples. The 117-kDa band identified in retinal extract was absent in brain, indicating tissue specificity (upper panel). R, retina; B, brain. E, expression of RHBDD2 in mouse embryonic eyes during development. Protein extracts (50 μg) were loaded into each lane of a 4–12% gel that was subjected to SDS-PAGE, and results were normalized to α-tubulin. F, Western blot of retinal extracts at different developmental times. The upper and middle blots correspond to the trimeric and monomeric forms of RHBDD2, respectively. Protein levels were normalized to β-actin.