Bcl 2

RIPA buffer (Solarbio, Shanghai, China) was used to lyse cells and a BCA kit (Beyotime, Shanghai, China) was used to quantify protein levels. The protein concentration was about 50 mg/mL, which was separated by 10% SDS-PAGE gel. β-actin (1:5000, AbMART, Shanghai, China, M2009) was used as a loading control. The antibody Fam98b (1:2000, FineTest, Wuhan, China, FNab03001), α-SMA (1:2000, Abcam, Cambridge, UK, ab124964), Collagen I (1:2000, Abcam, Cambridge, UK, sc-59772), Smad2/3 (1:2000, Abcam, Cambridge, UK, ab63672), BAD (1:2000, AbMART, Shanghai, China, 67830-1-Ig), BAX (1:2000, AbMART, Shanghai, China, T40051), Bcl2 (1:2000, AbMART, Shanghai, China, T40056), TGFβ (1:500, Wanleibio, Shenyang, China, WL02998), P-Smad2/3 (1:1000, Absin, Shanghai, China, abs131873), NOTCH3 (1:1000, Proteintech, Wuahn, China, 55114-1-AP) were used as primary antibody. The secondary antibodies were obtained from Invitrogen (1:20,000, Thermofisher, SA5-35521, SA5-35571). An Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) was employed to scan the blots. The quantitative analysis of grey values was performed using Image J software (NIH, USA).

Total protein was extracted from cell lines using lysis buffer (Thermo Fisher Scientific), containing a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China), and quantified using the Bradford method. Next, 40 µg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (8%), and the separated proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with primary antibodies against: TRAF4 (H2818, 1:100, Santa Cruz Biotechnology), Eg5 (1:1000), Smurf2 (F0641, 1:100, Santa Cruz Biotechnology), HA (TA180128S, 1:1000, Origene), α-tubulin (ab52866, 1:1000, Abcam), Caspase-3 (M005851F, 1:1000, Abmart), Bcl-2 (T40056F, 1:1000, Abmart), Bax (T40051F, 1:1000, Abmart), Ki67 (550609, 1:1000, BD Pharmingen), GAPDH (AF0006, 1:2000, Beyotime) and β-actin (1:2000, Beyotime). Next, the membranes were incubated with secondary HRP-conjugated antibody, anti-mouse immunoglobulin G (IgG), or anti-rabbit immunoglobulin (1:2000, Santa Cruz Biotechnology) at 37°C for 2h. Finally, antibody binding wasvisualizedusing electro-chemiluminescence (Thermo Fisher Scientific), and quantified using ImageJ (National Institute of Health, Bethesda, MD, USA).

RAW264.7 cells were harvested at 24 h and 48 h post-infection and lysed in lysis buffer on ice for 40 min. The supernatant was obtained by centrifugation at 13,000 rpm for 20 min at 4 °C. Protein concentration was determined using the bicinchoninic acid assay. Total cellular protein was extracted by boiling lysates for 10 min in 5× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Proteins were separated by SDS-PAGE on a 12% polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked for 2 h at room temperature in Tris-buffered saline containing 0.5% Tween-20 (TBST) and 10% skimmed milk and then incubated overnight at 4 °C in blocking solution containing antibodies against Bax (1:2000; Abmart, Shanghai, China), Bcl2 (1:2000; Abmart), cytochrome C (1:5000; Abcam, Cambridge, MA, USA), Caspase-9 (1:1000; Proteintech, Wuhan, China), Caspase-3 (1:1000; Proteintech), or β-actin (1:1000; Proteintech). The membranes were washed four times with TBST for 8 min, incubated for 2 h with the corresponding HRP-conjugated secondary antibody (1:5000; Zhongshan Golden Bridge Biotechnology, Beijing, China), and washed four times in TBST for 8 min. The signal was visualized using an ECL chemiluminescence kit (Beyotime, P0018FS, Shanghai, China) and imaged by the Gel Image System (Tannon Biotech, Shanghai, China).