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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Mol Cancer Res. 2014 May 16;12(9):1267–1282. doi: 10.1158/1541-7786.MCR-13-0652-T

Figure 5.

Figure 5

The full-length GM-CSFRα subunit is detected in the cytosol and the nucleus of GM-CSFRα-transfected 293TF cells. (A) Schematic diagram of GFP-tagged full-length GM-CSFRα and its truncated extracellular and intracellular fragments. The white box denotes the membrane domain. (B) The full-length GM-CSFRα subunit and its extracellular and intracellular fragments were successfully transfected into 293TF cells. Whole-cell protein extract of 293TF cells transfected with GFP-tagged full-length and its truncated extracellular and intracellular fragments were immunoprecipitated with rabbit anti-GFP antibodies; the GFP-tagged protein was detected with mouse anti-GFP antibodies. As shown, full-length-tagged GM-CSFRα and its tagged extracellular and intracellular regions were detected. Differences in signal intensity may represent differences in protein expression and/or transfection efficiency. I.P., immunoprecipitate; B, beads (control). (C) Similarly, confocal microscopy studies detected GFP-tagged full-length GM-CSFRα and its extracellular and intracellular fragments in the cytosol and the nucleus of the transfected 293FT cells. (D) The transfected full-length GM-CSFRα subunit and its extracellular and intracellular regions were detected in the cytosol and the nucleus of 293TF cells. Western immunoblot analysis detected GFP-tagged cytoplasmic (S6-positive, lamin B-negative) and nuclear (lamin B-positive, S6-negative) full-length, as well as extracellular and intracellular constructs of transfected GM-CSFRα. (E) GM-CSFRα antibodies detect the extracellular but not the intracellular region of the GM-CSFRα subunit. (F) Cytosolic (C) and nuclear (N) GFP-tagged full-length GM-CSFRα and its extracellular domain pulled down by GFP antibodies, but not by beads (B; control), bound GM-CSFRα antibodies.