(A) Schematic depicting domains in FL SNX9 and constructs used for GST pull-down assays (left). Lysates from AKR-2B cells untreated (–) or stimulated (+) for 45 minutes with 5 ng/ml TGF-β were incubated with GST beads or the indicated fusion proteins immobilized on GST beads. Bound proteins were eluted and assessed by Western blot analysis for pSMAD3 or pSMAD2. Cell lysate reflects signal obtained from 10 μg total protein; the slower migrating band in the pSMAD3 lane is a nonspecific protein (right, representative of 3 separate experiments). (B) AKR-2B cells were transduced for 90 minutes with the indicated concentration of TAT peptide. Following washing and 1 hour TGF-β (5 ng/ml) treatment, nuclear fractions were isolated and assessed by Western blot analysis for pSMAD2, pSMAD3, or histone deacetylase 1 (HDAC) (left, representative of 3 separate experiments). Quantitation of nuclear pSMADs was performed with ImageJ software (NIH) and represents the mean ± SEM of 3 experiments (right). (C) Left panels: AKR-2B cells were incubated with vehicle (top panels) or transduced (bottom panels) as in B with TAT-SH3 or TAT-LC (1.8 μM). Following treatment with or without TGF-β (5 ng/ml) for 1 hour, immunofluorescence for SMAD3 or the HA-tagged TAT peptide was performed as described in Methods and nuclei were stained with DAPI. Upper and lower panels show 2 distinct microscopic fields for each condition. Original magnification in C: ×100. Right: quantitation of nuclear SMAD3 from 30 cells in each of 3 experiments. *P < 0.05; **P < 0.005; ***P < 0.0005, 1-way ANOVA followed by Dunnett’s multiple comparisons test.