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. 2011 Nov 11;6(11):e27478. doi: 10.1371/journal.pone.0027478

Figure 4. LAPTM4a and LAPTM4b bind Nedd4 via their PY motifs.

Figure 4

(A) LAPTM4a's binding to Nedd4 is dependent on its 2nd and 3rd PY motifs. A Co-IP was performed on whole cell lysates from Hek293T cells co-expressing V5-tagged Nedd4 and mCh-LAPTM4a (WT, 3PA, P208A, P213A or P228A). Nedd4 was precipitated using anti-V5 antibodies, samples were separated on SDS-PAGE and transferred to nitrocellulose. Anti-mCherry antibodies were used to detect binding of mCh-LAPTM4s to Nedd4. (B) LAPTM4b binding to Nedd4 is dependent on its PY motifs. A CoIP was performed on whole cell lysates from Hek293T cells co-expressing V5-tagged Nedd4 and mCh-LAPTM4a-WT, mCh-LAPTM4b-WT or mCh-LAPTM4b-2PA, as in (A).The housekeeping proteins Tubulin and GAPDH were used interchangeably as loading controls for the cell lysates. (IgG bands are detected due to the HRP-conjugated 2o antibody's specificity for mouse Ig, which detects the Ig heavy and light chains of the V5 antibody used during the pulldown.).