0.22 m pvdf membrane

The midbrain and PC12 cells were collected and lysed using RIPA buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.5% sodium dexoycholate, 1 mM PMSF and 10 µg/ml leupeptin). The lysate was centrifuged at 12,000g for 20 min at 4°C, and the supernatant was quantified with BCA reagents (Thermo, Rockford, IL, USA). Proteins (30 µg) were separated on a 12% gel and transferred onto a 0.22 µm PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% (w/v) non-fat milk (Bio-Rad) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 0.5 h at room temperature, and then the membranes were incubated overnight at 4°C with the following primary antibodies: TH (1:1000, Abcam), α-syn (1:1,000, Abcam), FGFR1 (1:1,000, CST), GRP78 (1:1,000, Abcam), CHOP (1:500, Proteintech), TRB3 (1:100, Santa Cruz Biotechnology), LC3A/B (1:1,000, CST), mTOR (1:1,000, CST), p-mTOR (1:1,000, CST), and p62 (1:1,000, CST). The membranes were washed with TBST three times and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Then, the signals were detected using the Chemi DocXRS + Imaging System (Bio-Rad), and the bands were quantified using densitometric measurement by the Quantity-One software. All experiments were repeated three times.

Proteins from whole-cell was extracted with 1% Triton X-100 lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations of the extracts were determined by the BCA assay kit. 40 μg total protein from each sample was separated by ExpressPlus PAGE Gel (10 × 8, 4–20%, 15 wells) (Genscript, Nanjing, China) and transferred onto 0.22 µm PVDF membrane (Bio-Rad, Hercules, CA, USA) using a semidry transfer system (Bio-Rad, USA). Blots were blocked at room temperature for 15 min in QuickBlock Blocking Buffer for Western Blot (Beyotime, Shanghai, China) on a shaker, and then incubated with primary antibodies specific to LYRM4 (1:2000) and β-actin (1:5000) overnight at 4 ºC, respectively. The membrane was washed in TBST for 3 × 10 min and then incubated with HRP-conjugated anti-rabbit (1:5000) and anti-mouse (1:20000) IgG secondary antibody at room temperature for 1.5 h while gently agitating. Signals were detected with SuperSignal West Femto maximum sensitivity substrate according to the manufacturer`s protocol.

Bat and human intestinal organoids were lysed and subjected to total RNA extraction using the RNeasy kit, followed by reverse transcription using the PrimeScript RT-PCR kit (Takara, PR014B) and an oligo(dT) primer. The resultant cDNAs were used to measure mRNA expression levels of cellular genes using LightCycler 480 SYBR Green I Master Mix (Roche) and an LC480 thermocycler (Roche). Data were analyzed by the delta-delta Ct method. qPCR primers were listed in Supplementary Table 2.
For Western blot, human intestinal organoids were lysed in RIPA buffer supplemented with protease inhibitors (Roche). The lysates were separated in 12% SDS-PAGE and then transferred to a 0.22 µm PVDF membrane (Bio-Rad). After overnight blocking with 5% skimmed milk (Bio-Rad), the membrane was incubated with an anti-ISG15 antibody (Thermo Fisher Scientific, MA5-15029) for 2 h at room temperature, followed by incubation with an HRP-conjugated secondary antibody and detection with immobilon crescendo western HRP substrate (Millipore). Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).