Pc101

Cells were lysed by radio immunoprecipitation assay lysis buffer (EpiZyme PC101) followed by centrifuging at 13 000 × g for 15 min at 4°C. Total protein was collected and stored at −80°C until use. The protein samples were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane that was blocked for 15 min with QuickBlock Blocking Buffer for Western Blot (Beyotime, Shanghai, China). They were then incubated overnight at 4°C with primary antibodies including: rabbit polyclonal antibody to IFITM1, IFITM2, IFITM3 (Proteintech; 11727-3-AP, 12769-1-AP, 11714-1-AP), IFITM5 (Abcam; ab230863) and β-actin (CST; 4970S), anti-ASFV p30 mouse monoclonal antibody (provided by our laboratory) and anti-ASFV p72 mouse monoclonal antibody (Zoonogen M100068), followed by a 1 h incubation with secondary antibodies: Alexa Fluor 790 conjugated goat anti-mouse polyclonal antibody (Abcam; ab186695) and Alexa Fluor 680 conjugated goat anti-rabbit polyclonal antibody (Abcam: ab186696). Protein bands were visualized by Odyssey Sa (LI-COR, Lincoln, NE, USA). WB quantification by densitometry for each viral proteins or SwIFITM proteins and beta-actin are expressed using the ImageJ package.

Normal endometrium tissues and ovarian endometriosis tissues were lysed in RIPA lysis buffer (PC101; EpiZyme, Shanghai, China) with protease and phosphatase inhibitor cocktail. The protein concentration was detected by a BCA protein assay. The same amounts of total protein (32 µg) were loaded and separated onto 12.5% SDS-PAGE and was then transferred to PVDF membranes. After blocking with protein-free rapid blocking buffer (PS108P; EpiZyme, Shanghai, China) for 0.5 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies rabbit anti-PDLIM3 antibody (1:2,000; Servicebio) was used and rabbit anti-β-Actin (1:5,000; ABclonal) served as the internal control.
The membranes were probed with secondary antibodies (1:3,000, Servicebio) for 1.5 h at room temperature and three washes in TBST were processed to remove excess secondary antibody. We then used an ECL western blotting kit (NCM Biotech, Suzhou, China) to visualize and image the targeted protein bands.

Lung tissue and cellular protein were extracted using RIPA buffer (EpiZyme, PC101, Shanghai, China) with 0.1% PMSF, lysed on ice for 30 min, and centrifuged at 12,000 r for 10 min. The protein was quantified using a BCA Kit, and a 30 μg protein sample was obtained for 12-15% SDS-PAGE electrophoresis and transferred onto the PVDF membrane and then probed with rabbit anti-ENO2 (Bioss, bs-6273R, Beijing, China), Rabbit anti-SP (Beyotime, AF8094, Shanghai, China), Rabbit anti-VIP (Beyotime, AF8331, Shanghai, China), or Rabbit anti-CGRP (Beyotime, AF6495, Shanghai, China) primary antibodies overnight at 4×C. The secondary antibody of horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin G (IgG) (Boster, BA1055, Wuhan, China) was diluted to 1 : 3,000. The signals were detected using ECL (Millipore, WBLUR0100, Millipore, USA). The bands were visualized with the chemiluminescence detection system (Tanon 5200 Multi, Beijing, China), and the protein expression levels were normalized to GAPDH levels. The integrated density of proteins was quantified using ImageJ software (NIH).

At 20 h after transfection, HEK293T cells transfected with pcDNA3.1, HA-REEP6, HA-REEP6 c.268G>C, and HA-REEP6 c.468delC were treated with 100 μM cycloheximide (CHX, HY-N0901, MedChemExpress, NJ, USA) dissolved in DMSO. Cells were scraped at 0, 7, and 10 h after CHX exposure using ice-cold PBS. Cells were centrifuged, and lysed by RIPA lysis buffer (PC101, EpiZyme, Shanghai, China) including 1% protease inhibitor cocktail (GRF101, EpiZyme). After brief sonication, cell lysate was collected. The protein concentration was determined with BCA protein assay kit (P0011, Beyotime, Shanghai, China). All samples were boiled for 5 min at 100 °C, diluted by 5X SDS loading buffer (LT101S, EpiZyme). Equal amounts of protein were loaded on a 12.5% polyacrylamide gel for SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore, Burlington, MA, USA). PVDF membranes were blocked in 5% skim milk prior to incubation with mouse anti-HA (1:500; 2367S, Cell Signaling Technology, Beverly, MA, USA) and rabbit anti-β-actin (1:1000; 4970S, Cell Signaling Technology). After 2 h of incubation with secondary antibodies at room temperature, the membranes were visualized on a Bio-Rad ChemiDoc MP Imaging System. β-Actin was used as loading control.

After anesthesia, mouse retinas were collected on ice as soon as possible and stored at − 80 °C. The retinas were immersed in 200 µL RIPA lysis buffer (PC101, EpiZyme, Shanghai, China) and ultrasonicated to obtain homogenates. The samples were centrifuged at 4 °C and 2000×g for 20 min, and the supernatant was collected. For analysis, samples were added to wells according to the instructions of the ELISA kit manufacturer. Then, 100 µL horseradish peroxidase (HRP)-labeled antibody was added to each well except the blank wells, and the plate was sealed with a sealing membrane and incubated for 60 min in a 37 °C incubator. After the liquid was discarded, the wells were washed with washing solution (350 µL per well) and the plate was allowed to rest for one minute; this process was repeated for a total of 5 times. Substrate A and substrate B (50 µL) were added to each well, and the plate was incubated at 37 °C for 15 min away from light. Finally, 50 µL of stop solution was added to each well, and the OD value of each well was measured at 450 nm within 15 min.

Total proteins were extracted from cells using RIPA lysis buffer (PC101, Epizyme, Shanghai, China) with protease inhibitor cocktail (GRF101, Epizyme, Shanghai, China). Equal amounts of proteins were transferred to polyvinylidene fluoride membranes and imaged on an Odyssey scanner (LI-COR Biosciences, USA) after incubation with primary ferritin heavy chain (FTH) (T57085, Abmart), ferritin light chain (FTL) (T56955, Abmart), and β-tubulin (M30109, Abmart) and secondary antibodies (#7054, #7056, Cell Signaling Technology). The primary antibodies were diluted at 1:1,000 and secondary antibodies were diluted at 1:2,000.