Af1027

Clinical LF specimens (non-LFH group:LFH group = 2:2) were snap-frozen in liquid nitrogen and stored at −80°C for Western blotting. Total protein from each LF specimen was extracted in RIPA lysis buffer (Santa Cruz) and quantified using the BCA assay (Pierce). After denaturation, proteins specimens were separated using gel electrophoresis on 8%–12% SDS-PAGE, and then were transferred onto PVDF membranes (Roche Applied Science, Indianapolis, IN, USA). Using 5% nonfat dry milk for 2 hours at room temperature, The membranes were blocked in 5% nonfat dry milk for 2 h and then incubated overnight at 4°C with the following primary antibodies: Beclin1 (1:500; AF5128, Affinity), P62 (1:500; AF5384, Affinity), FN1 (1:500; AF5335, Affinity), TGFβ1 (1:500; AF1027, Affinity), NGF (1:500; AF5172, Affinity), HMOX1 (1:500; AF5393, Affinity), CAT (1:500; DF7545, Affinity), SIRT1 (1:500; TU365233, Abmart), and GAPDH (1:5000; AP0063, Bioworld). After, the membranes were incubated with goat anti-rabbit IgG (H+L) HRP secondary antibody (1:5000; RM3002, Rayantibody) for 2 h at room temperature. The proteins bands were detected using an enhanced chemiluminescence kit (KF005, Affinity), and chemiluminescence signals were quantified with Image Lab statistical software (Bio-Rad, Hercules, CA, USA).

Pancreatic cancer tissues and adjacent normal pancreatic tissues were obtained from pancreatic cancer patients. The research protocols were approved by the Research Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (ID: 20210728-31). All participants gave written consent authorizing the analysis of their tissue samples and medical information for scientific research.
After dewaxing and rehydration, tissue sections were put in EDTA solution, and antigen recovery was performed at 100°C for 30 min. After natural cooling to room temperature, an endogenous peroxidase blocker was used to block endogenous peroxidase activity for 15 min. After washing twice in PBS, tissue sections were incubated with the primary antibody anti-ADAMTS12 (ab203102, Abcam); anti-CD 163 (ab182422, Abcam); or anti-TGF beta 1 (AF1027, Affinity), at 4°C overnight. The next day, the tissue sections were washed twice in PBS and were then incubated with a secondary antibody at room temperature for 1 h. The immunohistochemical reactions were visualized with 3,3,0-diaminobenzidine (DAB), and hematoxylin was used for nuclear staining in all tissue sections. The protein levels were evaluated based on the percentage of positive cells and staining intensity (0, negative; 1+, weak; 2+, moderate; 3+, strong).

To isolate the total proteins, U251 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime). The protein samples were segregated by 10% SDS-PAGE, which were then deposited onto polyvinylidene difluoride membranes. After that, the membranes were blocked with 5% nonfat milk for 1 h before being incubated with primary antibodies anti-TGF-β (1 : 2000, AF1027, Affinity, Cincinnati, OH, USA) or anti-Smad4 (1 : 2000, AF5247, Affinity) for 12 h at 4°C. GAPDH was regarded as an internal control (1 : 3000, AF7021, Affinity). After being washed with Tris-buffered saline with Tween 20 (TBST), the membranes were further treated with a secondary antibody (HRP-IgG, 1 : 2000, ab205718, Abcam, Cambridge, England) for 1 h at 25°C in the dark. An ECL kit (Pierce, Rockford, IL, USA) was used to visualize the protein bands, which were then photographed using a Gel imaging system (Tanon 3500, Shanghai, China).