FIGURE 6.

Protein/protein interaction between LIMP2/SCARB2 and PRDX6 in vitro and in vivo. A, co-immunoprecipitation of recombinant PRDX6-His₆ by recombinant protein A-His₆-LIMP2 is favored in buffer of pH 5 compared with buffer of pH 7 (lanes 8 and 9). Representative immunoblot from two separate experiments. Lane 1 demonstrates molecular weight markers visualized by scanning at 700 nm. Lanes 2–7 illustrate nonspecific interactions between IgG-Sepharose beads with protein A alone (lanes 2 and 3), protein A and PRDX6-His₆ (lanes 4 and 5), or PRDX6-His₆ alone (lanes 6 and 7), at pH 7 and 5. Lanes 10–12 contain input protein for PRDX6-His₆ and protein A-His₆-LIMP2. B, representative confocal images of the Duolink PLA interaction between mouse anti-PRDX6 and rabbit anti-LIMP2 (red puncta). For negative controls, mAT2 cells were incubated with mouse anti-PRDX6 alone. The nuclei are labeled with DAPI and differential interference contrast (DIC) images were captured to distinguish the lamellar bodies within mAT2 cells. The red puncta represent positive Duolink signals showing the interaction between PRDX6 and LIMP2; scale bars, 10 μm. Summary data are shown for average number of PLA products per AT2 cell and were derived from two separate experiments, each using lung epithelial cells isolated from four mice of each genotype with 20–25 single AT2 cell images analyzed per genotype per experiment for each of the conditions shown. The box and whiskers plot shows the median (center line), 75th and 25th percentiles (upper and lower limits of the box), and range of raw data (maximum to minimum as hinges). The data were analyzed by unpaired t test, which showed p < 0.0001 WT versus pearl using both antisera in the proximity ligation assay.