Figure 5

The tail domain of NSP2 induces autophagy by binding 14-3-3ε. A Immunofluorescence microscopy showing the co-localization of 14-3-3ε and NSP2 protein domains. The co-localization of truncated EGFP-tagged NSP2 plasmids (NSP2-GFP, OTU-GFP, HV-GFP, TM-GFP, and Tail-GFP) with 14-3-3ε was visualized in transfected 293T cells and Marc-145 cells by immunofluorescence microscopy. GFP-tagged proteins are shown in green and endogenous LC3 proteins are shown in red. Yellow in the merged images represents co-localization between proteins. Bar size: 25 µm. B The amino acid sequence of the NSP2 tail region containing the 14-3-3ε-binding motif, and the RTEP and KTAP (blue box to mark the location) tail mutants (top). 293T cells were transfected with an empty vector, wild-type Tail-GFP plasmids (WT) or mutant GFP-tagged tail plasmids (RTEP or KTAP). At 24 h after transfection, cell lysates were harvested and used to perform GFP pull-down assays using GFP-trap beads, followed by western blot using anti-14-3-3ε antibodies. C 293T cells were transfected with wild-type Tail-GFP plasmids (WT) and EGFP-tagged Tail-GFP mutant plasmids (RTEP or KTAP) for the indicated time points. Whole-cell lysates were collected and probed with anti-GFP, anti-p62, anti-LC3, and anti-β-actin (loading control) antibodies for western blot analyses.