Figure 1.

Dab2 is not essential for TGF-β receptor internalization, but it impacts TGF-β Smad signaling. (A) Serum-starved Dab2 WT cells or Dab2 KD clones 16 and 18 (the numbers represent different clones of NIH-3T3 cell lines expressing stably integrated chimeric GM-CSF TGF-β receptors) were either left untreated (−) or stimulated with 50 ng/ml GM-CSF (activates the chimeric receptors) or 5 ng/ml TGF-ß (activates the native receptors) for 45 min. Lysates were prepared from parallel plates and equivalent protein (30 μg) immunoblotted with a Dab2 and a phospho-specific Smad2 antibody (pSmad2). The blot was stripped and probed for total Smad2 and GAPDH to confirm loading. (B) Internalization of ¹²⁵I-labeled GM-CSF through chimeric TGF-β receptors was performed in WT and Dab2 KD clones (KD 16, 17, 18, and 50). The ratio of internalized versus cell surface receptors was determined at 1 and 2 h. The graph shows average pooled data of the four KD clones, whereas individual values of the KD clones are shown in the inset. The results represent the mean ± SD of three different experiments performed in duplicate. (C) Dab2 WT and Dab2 KD (clone 16) cells were prebound with radiolabeled antibody to the chimeric type II receptor (GMCSF2RB) and then incubated at 37°C for the indicated times. Surface antibody was removed by acid treatment at 4°C, after which cells were processed to determine internalized radioactivity (as described in methods). Internalized counts are expressed as percentage of total cell-associated radioactivity at the indicated times and represent the mean ± SD of two experiments done in duplicate. Inset. serum-starved Dab2 WT cells were either left untreated (−) or stimulated (+) with 50 ng/ml GM-CSF (lane 2) or anti-GM-CSF receptor β antibody (GMCSF2RB; 1 μg/ml lane 4; 10 μg/ml lane 6). Lysates were prepared and equivalent protein (25 μg) immunoblotted with a phospho-specific Smad2 antibody (pSmad2). The blot was stripped and probed for total Smad2.