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. 2023 Apr 1;299(5):104668. doi: 10.1016/j.jbc.2023.104668

Figure 2.

Figure 2

Colocalization of Hsp90α/β and SARS-CoV-2 Orf7a, Orf7b, Orf3, M, and N proteins.A, Western blot analysis of the interaction between SARS-CoV-2 Orf7a, Orf7a, Orf3, M, or N proteins with endogenous Hsp90α/β proteins by reverse coimmunoprecipitation (CoIP) assay. B, the functional domains of SARS-CoV-2 M and N proteins; N-terminal domain (NTD), transmembrane region (TM), and C-terminal domain (CTD). HEK293T cells were cotransfected with Hsp90β-flag and myc-tagged WT or truncated M and N expression vectors. CoIP assay was performed using an anti-flag antibody. Colocalization of Hsp90α (C) or Hsp90β (D) with SARS-CoV-2 Orf7a, Orf7b, Orf3, M, N, and NSP15 proteins. HEK293T cells were grown on coverslips and then transfected with Hsp90β-flag or Hsp90α-flag vectors and myc-tagged SARS-CoV-2 protein vectors. At 24 h post-transfection, cells were fixed, permeabilized, and incubated with primary antibodies directed against flag (Hsp90α/β) and myc (SARS-CoV-2 Orf7a, Orf7b, Orf3, M, and N proteins) tags, followed by incubation with appropriate secondary antibodies. Cell nuclei were subsequently stained with DAPI. Images were collected on a confocal laser scanning microscope. Hsp90, heat shock protein 90; NSP, nonstructural protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.