FIGURE 1.

Identification of HSPA8 as a target membrane molecule for IBV Spike protein. (A) Determination of viral protein expression level of IBV M41 strain in CEK cells. CEK cells were infected with IBV M41 (TCID₅₀ = 10^6.5/ml, 50 μl/well) in 6-well plates. Infected cells were harvested at 0, 12, 24, 36, and 48 h post-infection. The whole cell lysates were subjected to SDS-PAGE and Western blot analysis with mAb 1H1 to spike protein. The expression of cellular actin protein was used as control. (B) Silver staining of membrane proteins of CEK cells after immunoprecipitation assay. Membrane protein extracts of IBV M41 infected CEK cells were immunoprecipitated with mAb 1H1 (lane 3) to IBV M41 spike protein, and mouse IgG (lane 2). Lane 1, protein marker. Arrow indicated the protein band at 70kDa specifically precipitated by mAb 1H1. (C) Western blot analysis of membrane proteins of CEK cells after immunoprecipitation assay. Sample orders were same as shown in (B). (D) Amino acid sequence of HSPA8. The gel fragment indicated with the arrowhead in (B) was analyzed by LC-MS/MS. The resulting peptide sequences of HSPA8 were underlined and marked as red color. Full length HSPA8 sequence was searched against chicken protein database in Uniprot. (E) Western blot analysis of HSPA8 present in the membrane protein extracted from CEK cells. Membrane protein fraction of CEK cells and whole cell lysates of CEK cells were immunoblotted with antibodies against the cytoplasmic protein marker (GAPDH), nuclear protein marker (Histone3), membrane protein marker (HSP90AB1) and HSPA8. WCL, whole cell lysates.