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. 2021 Dec 15;10(12):1628. doi: 10.3390/pathogens10121628

Figure 4.

Figure 4

Identification of the MbovP0145-interacting protein by GST pulldown and confirmation of β-actin interaction with MbovP0145. (A) MbovP0145-interacting protein by GST pulldown. Two differential protein bands (indicated by (A,B)) were discovered by comparison with the control samples. Two bands were excised from the gel and identified by MS. (B) Coimmunoprecipitation of MbovP0145 with β-actin. HEK293T cells were co-transfected with 3× Flag-actin and HA-MbovP0145, and the whole-cell lysates obtained at 36 h post-transfection were immunoprecipitated (IP) with anti-HA mAb. After separation by SDS-PAGE, MbovP0145 and β-actin were then detected with Western blotting assays using antibodies against either the HA or Flag tag. The identities of the protein bands are indicated on the right. (C) Colocalization of the exogenous rMbovP0145 protein (green) and β-actin (red). EBL cells were treated with the rMbovP0145 protein for 24 h. Cells were fixed and subjected to indirect immunofluorescence to detect MbovP0145 (green) and β-actin (red) with mouse anti-MbovP0145 and rabbit anti-β-actin antibodies, respectively. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. (D) Colocalization indicated that pEGFP-C1-MbovP145 transfection could co-locate with β-actin in HEK293T cells. The scale bars in the figure represent 20 μm.