Phospho c jun ser63

Animals were euthanized by cervical dislocation, and the lung and heart were prepared for histopathological analysis as previously described (24 (link)). In short, excised lungs were incubated in ice-cold endotoxin-free PBS, then in 10% NBF overnight, and then in 70% industrial methylated spirit until further processing. Paraffin-embedded 4 μm sections were stained with the following antibodies: TTF-1 (ab40880, Abcam), LacZ (AB9361, Abcam), c-Jun (610326, BD Biosciences), phospho–c-Jun Ser63 (9261, NEB), phospho–c-Jun Ser73 (9164, NEB), JunD (sc74, Santa Cruz Biotechnology Inc.), HMGA2 (8179, Cell Signaling Technology [CST]), and Ki67 (M7248, DAKO).
For quantification of tumor burden, the area (μm²) of existing individual tumors in each lobe was measured with QuPath software (40 (link)) (Measure > Show Annotation Measurements Tool) and represented as a percentage of tumor area per lobe. The analysis was performed uniformity across all lung sections, and the whole lungs were used to derive data. For quantification of cell proliferation, the number of Ki67⁺ cells was automatically counted in individual tumors with QuPath software (Cell Analysis > Positive Cell Detection Tool) and represented as a percentage of total cells in tumor area.

For protein analyses of lung tumor cell lines, cells were harvested, washed in PBS, and resuspended in cell lysis buffer (9803, NEB) supplemented with protease inhibitor cocktail (MilliporeSigma). Protein amount was measured by Lowry assay. The following antibodies were used for Western blot analyses: c-Jun (610326, BD Biosciences), phospho–c-Jun Ser63 (9261, New England Biolabs [NEB]), phospho–c-Jun Ser73 (9164, NEB), JunD (sc74, Santa Cruz Biotechnology Inc.), JunD (5000, NEB), phospho–SAPK/JNK Thr183/Tyr185 (4668 and 9251, NEB), SAPK/JNK (9252, NEB), phospho–p38 MAPK Thr180/Tyr182 (9211, NEB), p38 MAPK (9212, NEB), tubulin (ab7291, Abcam), and β-actin (ab49900, Abcam). All primary antibodies were used at 1:1000 dilution. Horseradish peroxidase–conjugated (HRP-conjugated) secondary antibodies were used at 1:5000 dilution, and anti–β-actin HRP antibody was used at 1:30,000 dilution. For Western blot utilizing the LI-COR system, DyLight goat anti–mouse 680 (5470, NEB) and DyLight goat anti–rabbit 800 (5151, NEB) secondary antibodies were used at 1:15,000 dilution. Separate immunoblots were used for each antibody.