Kim 1

Paraffin-embedded tissue sections from healthy control and MHV-1 infected group (10 microns) were incubated with specific antibodies to EGFR (cat# 05-1047 monoclonal antibody, Millipore Sigma, Burlington, MA, USA); TLR-2 (cat# 06-1119 polyclonal antibody, Millipore Sigma); HIF-1α (cat# H1Alpha67, ab1 monoclonal antibody, Abcam, Cambridge, UK); KIM-1 (cat# A2831 polyclonal antibody, ABclonal, Woburn, MA, USA); YKL-40 (cat# A20792 polyclonal antibody, ABclonal); MMP-7 (cat# AF907 polyclonal antibody, R&D systems, Minneapolis, MN, USA). All were used at 1:500 dilution. The sections were then incubated with respective secondary antibodies (Alexa flour 488 or Alexa flour 546 goat anti-rat, mouse, or rabbit IgG (Life Technologies, San Diego, CA, USA) at a concentration of 1:250 to 1:500. Fluorescent images from these sections were randomly collected with Zeiss LSM510/UV Axiovert 200 M confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) and quantified using Velocity 6.0 High-Performance Cellular Imaging Software (PerkinElmer, Waltham, MA, USA) as described previously, and normalized to the number of DAPI-positive cells, as well as to the area and intensity of DAPI.

Kidney tissues from healthy control and MHV-1-infected group were subjected to gel electrophoresis and immunoblotting as described previously [18 (link),19 (link),21 (link),38 (link)]. Briefly, equal amounts of proteins from uninfected, infected, and drug-treated mice kidney tissue extracts were subjected to SDS–polyacrylamide gel electrophoresis and then electrophoretically transferred. The membranes were then incubated with primary antibodies to EGFR (cat# 05-1047 monoclonal antibody, Millipore Sigma, Burlington, MA, USA); TLR-2 (cat# 06-1119 polyclonal antibody, Millipore Sigma); HIF-1α (cat# H1Alpha67, ab1 monoclonal antibody, Abcam, Cambridge, UK); KIM-1 (cat# A2831 polyclonal antibody, ABclonal, Woburn, MA, USA); YKL-40 (cat# A20792 polyclonal antibody, ABclonal); MMP-7 (cat# AF907 polyclonal antibody, R&D systems, Minneapolis, MN, USA). All were used at 1:2,000 dilution. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Vector Laboratories, Newark, CA, USA) were used at 1:5000 dilution. The optical density of the bands was measured with the Sigma Scan Pro program (Jandell Scientific, CA, USA) as a proportion of the signal of a housekeeping protein band (β-actin) [38 (link)] (Table S1).