Chemiluminescent hrp substrate

The protein expression levels of STAT3, EGFR, BCL2L1 and CASP9 in ECA-109 cells were measured by western blot analysis. Specifically, ELE-treated ECA-109 cells were collected and total cell proteins were extracted in RIPA buffer (Beyotime, Shanghai, China) for 30 min on ice. Protein concentrations were measured using a BCA protein assay kit (cat. no. P0012; Beyotime, Shanghai, China) according to the manufacturer’s instructions. The proteins were resolved by SDS loading buffer and denatured at 95 °C for 5 min. Subsequently, a total of 30 μg of proteins were separated by SDS-PAGE on 10% gels, which were then transferred onto PVDF membranes and blocked with 5% skim milk for 2 h at room temperature. The PVDF membranes were incubated with the corresponding primary antibodies (STAT3, EGFR, BCL2L1 and CASP9 (ZEN BIO, China); GAPDH (Proteintech, USA)) overnight at 4 °C. After washing with TBST, the PVDF membranes were incubated with secondary antibody overnight at room temperature. Finally, detection was routinely performed with a chemiluminescent HRP substrate (Beyotime, Shanghai, China) and an ECL imaging system (Tanon, Shanghai, China). The result of gels images was cropped and full-length gels and blots are included in the Supplementary Data.

Total protein was prepared with RIPA buffer (Beyotime, Shanghai, China) and protein concentration was determined using a BCA kit (Beyotime, Shanghai, China). Thirty µg of protein were used in western blot analysis according to our previous study 18 (link). The rabbit anti-human monoclonal H3K4 antibody (#9751, 1:1000 dilution, Cell Signaling Technology) and a mouse anti‑human monoclonal histone 3 antibody (1:1000 dilution, Beyotime, China) were employed in our study. Detection was routinely performed with a chemiluminescent HRP substrate (Beyotime, Shanghai, China) and an electrogenerated chemiluminescence (ECL) imaging system (Tanon, Shanghai, China).

Equal amount of 20 μg salivary proteins from different groups were applied to the 10% SDS-PAGE and transferred from gels to the PVDF membrane as described in 2.4. After being blocked with 5% skimmed milk in TBST for 1 h, the membrane was incubated with primary antibody of mouse monoclonal anti-human MUC5B (1:1000 in TBST, Abcam, Ab105460) or rabbit monoclonal anti-human immunoglobulin A (IgA) (1:1000 in TBST, Abcam, Ab124716) at 4 °C overnight. The membrane was then washed three times with TBST and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The goat anti-mouse Immunoglobulin G heavy and light chains (IgG H&L) for MUC5B (1:5000 in TBST; Abcam, Ab205719) and the goat anti-rabbit IgG H&L for IgA (1:5000 in TBST, Abcam, Ab205718) were used, respectively. After being washed three times with TBST, the membranes were finally detected by using the chemiluminescent HRP substrate (Beyotime, Haimen, China) and scanned with the Tannon 5200 Imaging System (Tanon, Shanghai, China).