Fig. 5.

LpUVOps1 is detected in membranes from VEs and MEs. (A) Chemiluminescent images of western blots of LpUVOps1 and LpOps5 antigens (1 μg per lane) probed with antibodies directed against these antigens. The blot was probed first with anti-LpUVOps1, stripped, and re-probed with anti-LpOps5. The antibody used is indicated on the left; the antigens are identified above the blots. Each primary antibody was diluted 1:1000; secondary antibodies were diluted 1:30,000. Each primary antibody labeled only the antigen against which it was generated. (B) Chemiluminescent images of western blots of membrane preparations from median eyes (ME), lateral eyes (LE) and ventral eyes (VE) probed with anti-LpUVOps1 and anti-LpOps5. Solubilized membranes from 2.5 VE nerves, 0.3 LEs and 0.5 MEs were loaded. Blots were immunostained for LpUVOps1 (1:50 dilution), stripped, then immunostained for LpOps5 (1:250 dilution). Anti-LpUVOps1 stained a single 32 kDa band from ME and VE membranes. No LpUVOps1-ir was detected in LE membranes. A doublet of LpOps5-ir at ~32 and 35 kDa was detected in membranes from LE and VE, but not ME. (C) Chemiluminescent images of western blots of ME and VE membranes probed with anti-LpUVOps1 (control, left lanes) and anti-LpUVOps1 that had been pre-absorbed with antigen (pre-abs, right lanes). Membranes were run on a single SDS gel separated by a lane of molecular mass markers. After proteins were transferred to nitrocellulose, the blot was cut so half the marker lane between the sample lanes was retained with each sample lane to allow the samples lanes to be aligned after immunostaining. Membranes from 1 ME were loaded on each lane and probed with either anti-LpUVOps1 or pre-absorbed anti-LpUVOps1 diluted 1:1000. Membranes from two VE nerves were loaded on each lane and probed with either anti LpUVOps1 or absorbed anti-LpUVOps1 diluted 1:50. Lanes probed with anti-LpUVOps1 each showed a clear band at approximately 32 kDa that was absent in lanes probed with pre-absorbed anti-LpUVOps1.