Fig 1: Effects of U18666A on PRV1028 production and protein expression. (A) PK-15 cells were infected with PRV1028, a recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion, at an MOI of 5 in the presence of U18666A (0.625–10 µg/mL) or DMSO (0.1%, v/v) as a control. At 48 h post-infection, virus titer in the culture supernatant was measured by plaque assay. Virus titer is presented in plaque-forming units (PFU)/mL. Data are mean ± SD (n = 3). *** p < 0.001. (B) PK-15 cells were infected with PRV 1028 at an MOI of 5 in the presence of U18666A (5 µg/mL) or DMSO (0.1%, v/v) as a control. At the indicated times post-infection, virus titer in the culture supernatant was measured by plaque assay. Data are mean ± SD (n = 3). (C) Effects of U18666A on the PRV 1028 VP26-mCherry protein expression. PK-15 cells were infected with PRV 1028 (MOI = 5) in the presence of U18666A (5 µg/mL) or DMSO (0.1%, v/v) as a control. Cell extracts were harvested at 24 h post-infection and subjected to WES protein analysis (ProteinSimple, San Jose, CA, USA) using rabbit anti-mCherry antibody (Abcam ab183628). Probing with anti-GAPDH antibody (Cell Signaling #5174) was included as a loading control. A mock-infected sample was included as a negative control. The asterisk (*) indicates the position of the degradation product from VP26-mCherry. A full image of WES protein analysis can be found in Supplementary Materials. Data are representative of three independent experiments. (D) The intensity of protein bands for VP26-mCherry and GAPDH was quantitated using the WES protein analysis software (ProteinSimple), and the ratio of VP26-mCherry/GAPDH was determined. Data are mean ± SD (n = 3) from three replicate runs in a representative experiment.
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