Fig. 3

Inverse correlation of Smurf2 and pHSP27 during RIPF. A L132 cells were transfected with either non-phosphorylated alanine (HSP27S15A/S78A/S82A; AAA) plasmids or phosphomimetic aspartate (HSP27S15D/S78D/S82D; DDD) plasmids and treated with 10 μg/mL of cycloheximide (CHX) for various time periods. B Confirmation of ubiquitin-related genes by quantitative RT-PCR using lungs from individual mice. C Smurf2 was stained for immunofluorescence (green). The graph shows Smurf2 expression of a directly irradiated region. Magnification, 50 × . Scale bar, 200 μm. D Western blots using cell lysates 12 h after 10 Gy IR in L132 cells, displayed by representative blot and quantifications. E Lysates of L132 cell lines with an empty vector or Smurf2 WT (Smurf2) 24 h after 10 Gy IR, shown by representative blot and quantifications. F Western blots using cell lysates 24 h after 10 Gy IR in L132 cells with siControl or siSmurf2 transfection. Ratio of each protein to β-actin in all western blot data (n ≥ 3). Biologically independent samples and results are representative of independent experiments (D-F). Data are expressed as mean ± SD. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. Control; ^#P < 0.05 and ^##P < 0.01 vs. Control-IR; ^†P < 0.05 and ^††P < 0.01 vs. treated and non-IR. G Proximity ligation assay (PLA) confirms endogenous AAA/DDD (Flag): Smurf2 interaction. Interactions with target proteins are indicated as red dots. Cell nuclei were counterstained with Hoechst (blue). Scale bar 10 μm. ^***P < 0.001 vs. AAA (n ≥ 3, mean ± SD). H Immunofluorescence staining for Smurf2 (green) and pHSP25 (red) in RIPF mouse lungs at 6 weeks after 75 Gy IR. Magnification, 200 × , Scale bar, 50 μm. (n ≥ 3, mean ± SD)