Fig. 3.

In vivo characterization of CrNFκB and CrIκB. (A) Hemocyte extracts were used for EMSA. Approximately 20 μg of extract was incubated with the CrFC κB probe (lanes 1 and 3–9) or mutant (Mut) CrFC κB probe (lane 2). Binding complexes were progressively abolished by CrIκB (lanes 3–6), although it remained unaffected by GST (lane 7). Helenalin (lane 8) decreases the intensity of the binding compared to DMSO (lane 9). (B Left) Supershift of the κB-binding complex by the anti-CrNFκB antibody. (B Right) Naïve and 1- and 3-hpi Pseudomonas-challenged horseshoe crab hemocyte nuclear extracts were incubated with the CrFC κB probe. (C) Degradation of CrIκB after bacteria infection. Hemocyte extracts were prepared from naïve and infected animals according to Ding et al. (30). Western blots show proteins extracted from hemocytes over time (min) of infection. Equal protein loading and transfer were verified by using Limulus actin. (D) Expression of CrNFκB, CrIκB, and CrFC. The hemocytes were collected 1–72 hpi. The Limulus actin 11 gene was the internal control. (E and F) CrNFκB affects the expression of immune-related genes. One hour after treatment with DMSO (♦), helenalin (■), MG-132 (•), or JSH-23 (▴), the horseshoe crabs were either left unstimulated (0 h) or challenged with P. aeruginosa, and the hemocytes were collected at the indicated hpi. Results are expressed as relative fold increase as compared with naïve control (0 h), which was set to 1. Without infection, the expression of CriNOS was undetectable. Thus, the expression level of CriNOS at 3 hpi was set to 1.