Figure 2: PRRSV co-located with MYH9 in the cells at early stage of PRRSV infection.

MARC-145 cells (a) and PAM cells (b) were infected with PRRSV at an MOI of 100 at 4 °C for 1 hr, followed by a temperature shift to 37 °C for 0, 5, 15, 30 mins and 1, 2, 4, 6, 8, 12 hrs and then fixed, stained with rabbit anti-MYH9 and pig anti-PRRSV antibodies, and analyzed by confocal microscopy. The location of MYH9 (red) and PRRSV (green) in MARC-145 cells and PAMs at 4, 6, 8 and 12 hrs was shown in Supplementary Fig. 4a,b. Scale bars, 10 μm. (c) MYH9 mRNA relative level in PRRSV infected and mock infected MARC-145 cells and PAMs was tested by quantitative RT-PCR. The data was normalized to MYH9 mRNA level in mock infected cells at 37 °C for 0 hrs. Error bars, mean ± s.e.m. (n = 3). (d) MYH9 protein level in PRRSV infected and mock infected MARC-145 cells and PAMs was tested by Western blot using the indicated antibodies. The samples from left to right were PRRSV mock infected and infected cells lysates. The α-tubulin was tested as a loading control. (e) After PRRSV binding to MARC-145 cells at 4 °C for 1 hr, the cells were shifted to 37 °C for 0, 5, 15, and 30 min and fixed without permeabilization. MYH9 (red) localization was visualized by confocal microscopy after staining with specific antibodies. PRRSV pre-mixed with pig anti-PRRSV serum before infecting MARC-145 cells, and mock infected MARC-145 cells were used as the parallel controls. Nuclei (blue) were stained with DAPI. Scale bars, 10 μm.