Ffpe total protein extraction kit

Proteins from FFPE samples were extracted using the FFPE Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) following the manufacturer's instruction. Then samples were precipitated with 4 × (v/v) ice-cold acetone and incubated at – 20 ℃ overnight. The precipitated proteins were centrifuged at 10,000×g at 4℃ for 15 min and air-dried.

Total protein was extracted from paraformaldehyde-fixed, paraffin-embedded pancreatic tissues using the FFPE Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) or from cultured PSCs using RIPA Lysis Buffer (Beyotime). Protein concentrations were determined using an enhanced BCA protein assay kit (Beyotime). The proteins (20 μg) were resolved by SDS-PAGE and blotted onto polyvinylidene fluoride membranes. The membranes were blocked in QuickBlock™ Blocking Buffer for 15 min (Beyotime) and then probed with primary antibodies against fibronectin, collagen I, GFAP, α-SMA, TGFβ1, SMAD3, phospho-Smad3 (Thr8), SMAD4, α-tubulin, and β-actin (Supplementary Table 2). HRP-conjugated goat anti-mouse IgG or anti-rabbit IgG (Boster, Wuhan, China) was used as the secondary antibody. Protein band intensities were quantified using ImageJ software (version 1.60).

IF was performed on FFPE sections after heat-induced antigen retrieval. Briefly, sections were blocked for 1 h, incubated overnight at 4°C with a rabbit primary antibody against human CACNG4 (Immunoway, YM3404), and subsequently incubated with a secondary Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Abcam, ab175696) for 1 h. Slides were mounted with mounting medium containing DAPI and imaged by a Leica SP8 laser scanning confocal microscope system. For WB, samples were pooled and extracted using the FFPE Total Protein Extraction Kit (Sangon Biotech, C500058) and then incubated with primary antibody against CACNG4 or β-actin (Cell Signaling, #3700). The proteins were visualized using HRP-conjugated secondary antibody (Cell Signaling, #7040 or #7076) and chemiluminescent HRP substrate (Bio-Rad).