Supplementary Figure 1. A. Immuno localization of PG and ANX-II in distal colonic crypts of Fabp-PG
mice. The colonic crypts were processed for staining with either PG or ANX-II-antibodies, respectively, as described in Fig 3. As described in the legend of Fig 3, the relative levels of PG retained within the distal (D) colonic crypts of transgenic mice were higher in 10-20% of the mice for unknown reasons. Representative data from one such mouse sample is presented in Supplementary Fig 1A. As can be seen from this example, relatively higher levels of PG and even ANX-II were present in the distal (D) crypts of this Tg mouse. Co-localization of PG and ANX-II in the merged images remained at the apical and lateral membranes of the crypts, with insignificant co-localization intracellularly. However, >80% of the Tg mice demonstrated significantly lower retention of PG in the distal colonic crypts as shown in Fig 3. B. Co-immunoprecipitation of PG with ANX-II in distal and proximal colonic crypts of Fabp-PG mice. Colonic crypts were isolated from the distal (D) and proximal (P) crypts of the Fabp-PG mice and processed for preparing cellular lysates as described in Methods. The cellular lysates (~600ug of protein) were precleared with normal rabbit serum + protein-A beads (Pierce) (~100ug in RIPA buffer), and gently agitated for 1h at 4°C. The lysates were centrifuged at 14,000xg for 10min at 4°C and the cleared supernatant used. 5μl of PG specific anti-polyclonal-Ab was added and incubated at 4°C overnight. The immune complexes were subjected to a pull-down assay with protein A Sepharose beads at 4°C for 5-6h. The beads were recovered by centrifugation at 14,000xg for 10min followed by extensive washes with 1ml RIPA buffer (10min x3). The beads were boiled in 1X SDS sample buffer and run on 12% SDS-PAGE followed by Western blotting with antibodies to either ANX-II or PG. Representative Western blots of co-immunoprecipitated (IP) ANX-II and PG from P and D colons of Tg mice are presented in lanes 2 and 4 as IP proteins. Inp = total amount of ANX-II and PG present in an equivalent volume of pre-cleared lysate sample, before subjecting the sample to IP. Data from four separate measurements from four mouse samples are shown as bar graphs in the lower panels. The filled bars represent the relative amounts of ANX-II and PG in the pre-cleared lysates (as input) before IP. The clear bars represent the relative levels of ANX-II and PG in the co-immunoprecipitated samples. Each bar = mean ± SEM of four separate measurements from four mice. * = p<0.05 vs. the corresponding levels in proximal colonic crypts. As a control experiment, cellular lysates from distal and proximal colonic crypts of WT mice were also processed for possible co-IP with Annexin II. Since, PG is not expressed in the colonic crypts of WT mice, samples from WT colonic crypts were negative for both PG and ANX-II. These results once again confirmed that the anti-PG-antibody, used in the current studies, was specific and did not detect any other protein either by IHC (Fig 3) or by Western Blot analysis (data not shown).
Supplementary Figure 2. Phosphorylation and nuclear translocation of p44/42 in vivo. A. Total crypt cellular and nuclear extracts prepared from distaldistal distaland proximal proximalcolonic crypts of wild type (WT) and Fabp-PG (Tg) mice were probed with antibodiesAbs against for phospho-ERK1/2 (p-p44/42) and total ERK1/2 (p44/42). The cellular pp44/42 level did not differ in distaldistal colons of WT and Fabp-PG mice (data not shown). distal colon. Proximal Proximalcolons, however, exhibited significant increases in pp44/42 level when the relative levels of phosphorylated kinase were corrected for the total levels of the corresponding kinase (Ai). Nuclear extracts also also exhibited significant nuclear accumulation of pp44/42 in samples from Fabp-PG vs. WT mice (Ai). The signal intensity of the bands in the Western blot was densitometrically analyzed. The densitometric readings for pp44/42 vs. total p44/42 for WT samples were arbitrarily assigned a 100% value. Ratios representing % change are shown as bar graph (Aii). Data in each bar graph represent Mean + SEM of 3 blots from 3 separate mice. * = P<0.05 vs. WT values. compared to their WT counterparts.B. IHC inof the frozen sections prepared from the proximal proximal(Pcolons of Fabp-PG mice exhibited significant enrichment inof pp44/42 in the nucleus throughout the longitudinal crypt axis (arrows), compared to distaldistal distalcounterpart, while distaldistal distalandor proximal proximalcolons from WT animals exhibited predominantly cytoplasmic and occasional nuclear staining (n = 3; barm).
Supplementary Figure 3. Immunohistochemistry of frozen sections with non-immune IgG. Frozen sections prepared from distal (D) and proximal (P) colons of wild type (WT) and transgenic (Fabp-PG) mice were immunostained with non-immune mouse IgG and analyzed by light microscopy. Representative sections from D and P colons are shown (n = 3; bar = 100m). Panel A represents an IgG control for IB staining shown in Figure 4D while panel B represents non-immune IgG control for phospho-p65-Ser276 staining shown in Figure 5Di. Neither control exhibited any specific staining with non-immune IgG.
Supplementary Figure 4. Immunohistochemistry of frozen sections with antibody to p65 phosphorylated at Ser 276 (pp65276). Frozen sections prepared from distal (D) and proximal (P) colons of Fabp-PG mice were immunostained with anti pp65276 and analyzed by light microscopy. Significant nuclear staining for pp65276 in the P colon of Fabp-PG mice (arrows) was observed, compared to D colon (upper panel; 200X; n = 3) which paralleled the staining pattern shown in Figure 5Di. Lower panel represents the counterstaining with hematoxylin to label the nuclei (200X; n =3).