Ab233706

Total protein was extracted from the different groups
using RIPA lysis buffer, and the protein concentrations
were measured using a BCA Protein Assay kit following
the manufacturer’s instructions. The protein samples (20
µg) were separated by SDS-PAGE and transferred to PVDF
membranes. After blocking with 5% non-fat milk at 37˚C for
one hour, the membranes were incubated at 4˚C overnight
with primary antibodies for TGF-β (1:1000, Abcam,
ab179695, USA), TNF-α (1:1000, Abcam, ab6671, USA),
TNF-β (1:1000, Abcam, ab227929, USA), NF-kB (1:1000,
Abcam, ab16502, USA), IL-1β (1:1000, Abcam, ab2105,
USA), IL-6 (1:1000, Abcam, ab233706, USA) and GAPDH
(1:1000, Proteintech, 60004-1-Ig, USA). After washing three
times, the membranes were then incubated with goat anti-rabbit IgG (1: 1000) for 2 hours at 37˚C. The protein bands
were visualized using the ECL system.

H&E, IHC, and IF staining were performed in PDAC tissues from archived formalin-fixed paraffin-embedded (FFPE) tumors and were performed according to standard protocols. For IHC, the slides were incubated with PGP9.5 (Servicebio, GB11159, 1:500), p75NRT (Abcam, ab52987, 1:100), GFAP (Proteintech, 16825, 1:200), S100β (Servicebio, GB11359, 1:150), IL-6 (Abcam, ab9324, 1:50), α-SMA (Abcam, ab5694, 1:200), N-Cadherin (Proteintech, 22018-1-AP, 1:100), Fibronectin (Proteintech, 15613-1-ap, 1:200), FAP (Cell signaling technology [CST], 66562,1:100) followed by HRP conjugated Goat Anti-Mouse IgG (H + L) (Servicebio, GB23301, 1:200) or HRP conjugated Goat Anti-Rabbit IgG (H + L) (Servicebio, GB23303, 1:200) and diaminobenzidine (Brown) (DAB, Dako). The slides were counterstained with hematoxylin (Dako), which stains nuclei blue, contrasting with the brown of HRP-DAB. Fibroblast morphology was confirmed based on the two pathologists’ assessments. IF was performed according to the manufacturer’s instructions (Panovue, 10004100100). Briefly, slides were incubated with primary antibodies for S100β (Servicebio, GB11359, 1:500), p75NRT (Abcam, ab52987, 1:100), α-SMA (Abcam, ab5694, 1:200), IL-6 (Abcam, ab233706, 1:50), and IL-1α (Proteintech, 16765-1-AP, 1:50) overnight at 4 °C, and stained with HRP-labeled goat anti-mouse/rabbit IgG secondary antibodies.