Ab53881

Lung sections were probed with primary antibodies (against SPDEF (ab53881, Abcam), STAT6 (ab44718, Abcam), FOXA2 (ab108422, Abcam), MUC5AC (ab24071, Abcam), and MUC5B (ab77995, Abcam)) and detected using an SABC kit (SA1020, Boster) according to the manufacturers’ protocols. The stained tissues were evaluated under a digital pathology scanner (PRECICE 500B, Unic-tech). ImageJ and IHC Profiler were used for quantitative analysis. IHC score was calculated as follows: (Number of pixels in zone) × (Score of the zone)/Total number of pixels in the image. The proportion of nuclear-positive staining cells was scored for analysis as follows: high proportion = 4, moderate proportion = 3, low proportion = 2, and negative staining = 1.

Lung protein extraction was performed with tissue lysis buffer (RIPA buffer, P0013B, Beyotime; phosphatase inhibitor cocktail 2, P5726, phosphatase inhibitor cocktail 3, P0044, Sigma). Then, the protein concentration was determined using a BCA kit (23227, Thermo), and primary antibody solutions diluted with 2.5% BSA (Amresco) (ERK1/2: 1:2,000, CST, 9102S; p-ERK1/2: 1:2,000, CST, 9102S; p-NF-κB: 1:2,000, Abcam, ab28856; GAPDH: 1:8,000, Abcam, ab181602; SPDEF: ab53881, Abcam; STAT6: ab44718, Abcam; and FOXA2: ab108422) and secondary antibodies (goat anti-rabbit, 1:5000, Proteintech) were used. ImageJ software was used for grayscale analysis.

Western blotting was performed as described previously [21 (link)] by using the following primary antibodies: anti-AR antibody (ab9474; dilution, 1:3000), anti-PDEF antibody (ab53881; dilution, 1:1000; Abcam), anti-MAD1 antibody (ab175245; dilution, 1:3000), anti-MYC antibody (ab32072; dilution, 1:3000), anti-β-catenin antibody (ab32572; dilution, 1:3000; Abcam), anti-AKT antibody (sc-135,829; dilution, 1:3000; Santa Cruz Biotechnology), anti-phosphorylated AKT antibody (anti-p-AKT; sc-7985-R; dilution, 1:3000; Santa Cruz Biotechnology), anti-ERK antibody (sc-514,302; dilution, 1:3000; Santa Cruz Biotechnology), anti-phosphorylated ERK antibody (anti-p-ERK; sc-81,492; dilution, 1:3000; Santa Cruz Biotechnology) and anti-EGFR antibody (ab52894; dilution, 1:3000; Abcam).