Bovine serum albumin (bsa)

The prostatic tissue was ground in liquid nitrogen and the total protein was extracted using a protein extraction kit. Equal amounts (40 μg/lane) of proteins were separated by SDS–polyacrylamide gel electrophoresis; the resultant bands were transferred onto PVDF membranes. After being blocked in 5% bovine serum albumin for 1 h at room temperature, the membranes were incubated with antibodies against TGF-β1 (1:500; Proteintech Group), RhoA (1:5000; Proteintech Group), ROCK1 (1:500; Proteintech Group), and β-actin (1:1000; Affinity Biosciences) overnight at 4 °C. The membranes were washed thrice in TBST for 30 min, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Affinity Biosciences) for 1 h followed, by a further washing step for 30 min. Finally, the bands were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific). The data were normalized using β actin as an internal control. All samples were analyzed independently via three repetitions and the mean values were determined.

IFA was performed to detect the localization of SARS-CoV-2 nsp1, SARS-CoV-2 nsp1-K164A and SARS-CoV nsp1 in HEK-293T cells. To examine expression of α-CoV nsp1 in HEK-293T cells, cells (2×10⁵/well) seeded in 24-well plates (Nest) were transfected with the corresponding plasmids for 24 h. Cells harbouring the empty PCAGGS vector were used as a control. Briefly, cells were fixed with 4 % paraformaldehyde for 10 min and permeabilized with precooled Triton X-100 for 10 min at room temperature. After three washes, the cells were blocked with 5 % bovine serum albumin in PBS for 1 h at 37 °C and incubated with anti-HA antibodies (1 : 1000; Proteintech) for 1 h at 37 °C. After four washes with PBST, a goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 488 (1 : 2000; Thermo Fisher Scientific) was added for 40 min at 37 °C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) for 10 min at room temperature. The samples were mounted in PBS and imaged using an LSM 510 Meta confocal laser-scanning microscope (Carl Zeiss, Zena, Germany).

All operations in this section were performed at low temperature. To evaluate PMN and MN populations, processed CSF cells or PBLs were stained with 1 μg/ml Hoechst 33342 for 5 min, then fixed in PBS containing 1% paraformaldehyde, and checked through an Influx flow cytometer (BD). For cell immunostaining, processed CSF cells or PBLs were blocked for 15 min in PBS containing 0.5% bovine serum albumin (Sigma-Aldrich, A1933) and 3% FBS and then incubated for 30 min with anti-VSIG4 (4 μg/ml, Santa Cruz, sc-53977), anti-CD1C (2 μg/ml, NOVUS, NBP2-62220), or equal concentrations of isotype-matched control antibodies. After washing three times with PBS containing 3% FBS, the suspension was incubated with 2 μg/ml Alexafluor 488-conjugated goat anti-mouse antibody (4408S, Cell Signaling) for 30 min in PBS containing 0.5% bovine serum albumin and 10% goat serum (B900780, Proteintech) and then incubated for another 5 min with 1 μg/ml Hoechst 33342. The cells were washed three times and fixed in PBS containing 1% paraformaldehyde. Cellular fluorescence was measured using an Influx flow cytometer, and data were analyzed using FlowJo software.