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. 2012 Jun 20;7(6):e39505. doi: 10.1371/journal.pone.0039505

Figure 1. Nedd4 specifically interacts with MAK-V.

Figure 1

(A) Typical profile of proteins purified on anti-FLAG M2 affinity gel from PC12TetOn cells treated with doxycycline (+) to express MAK-V-FLAG protein or left untreated (-). Silver-stained 4–12% gel is shown with positions of protein molecular weight markers (MW) shown in kD on the left. Arrows marks MAK-V-FLAG protein (*) and Nedd4 protein co-purified with MAK-V-FLAG (**). (B) Samples from (A) (IP anti-FLAG) and aliquots of cell lysates prior to purification (Input) were stained with anti-Nedd4 (anti-Nedd4) or anti-FLAG (anti-FLAG) antibodies. (C) Lysate of PC12TetOn cells treated with doxycycline to express MAK-V-FLAG protein was incubated with Protein G Sepharose (no Ab) or Protein G Sepharose with immobilized anti-Nedd4 antibodies (Nedd4 Ab). Bound proteins were analyzed by Western blotting with anti-FLAG (anti-FLAG) or anti-Nedd4 (anti-Nedd4) antibodies. (D) Proteins were pulled down from lysate of PC12TetOn cells treated with doxycycline to express MAK-V-FLAG protein with GST (GST) or GST-Nedd4 (GST-Nedd4) proteins immobilized on glutathione Sepharose. MAK-V-FLAG protein was detected with anti-FLAG antibodies (anti-FLAG). To monitor GST protein loading of glutathione Sepharose, eluted proteins were stained with anti-GST antibodies (anti-GST). Arrowhead marks full-length GST-Nedd4 chimeric protein.