RNAi-mediated knockdown of Cct1 or Cct2 does not modulate FHV protein A accumulation or membrane association in S2 cells. (A) Schematic of FHV protein A expression vector with C-terminal HA tag. The RNA template produced from pS2FA-HA is optimized for translation by inclusion of a 5' leader sequence (L) and a 3' polyadenylation signal (An), but these alterations in addition to the C-terminal HA tag render the RNA template incompetent for viral RNA replication due to changes in essential 5' and 3' cis elements. (B) FHV protein A accumulation determined by quantitative immunoblotting and β-galactosidase activity determined by enzyme assay in S2 cells co-transfected with pS2FA-HA and pS2LacZ after RNAi-mediated knockdown of the indicated dsRNA targets. P-value < 0.05* or 0.005**. (C) S2 cells stably expressing either an empty vector (lane 1) or pS2FA-HA (lanes 2-13) were treated with dsRNA targeting LacZ (lanes 2-4), Cct1 (lanes 5-7), Cct2 (lanes 8-10), or both Cct1 and Cct2 (lanes 11-13) for 72 h, induced with 0.5 mM Cu2+ for 18 h, washed in PBS, and either lysed directly in SDS-PAGE buffer to obtain total fractions (T) or subjected to saponin-mediated permeabilization and differential centrifugation to obtain soluble (S) and pellet (P) fractions, which correspond to cytosolic and membrane protein fractions, respectively . Fractions were analyzed by SDS-PAGE and immunoblotting for protein A, the cytosolic protein tubulin, or the membrane protein porin.