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. 2012 Sep 27;7(9):e46072. doi: 10.1371/journal.pone.0046072

Figure 5. Subcellular fractionation: opposite Effects of E2 and Pg on Cdh1 protein levels compared to p27, p-p27(T187), Skp2, and Cks1; Pg induces binding of Cdh1 to APC.

Figure 5

A, E2; B, Pg, Cell fractionation and analysis of Cdh1, Skp2, Cks1, p27 proteins and p-p27(T187): Cells were either untreated, treated with E2, E2 plus Lac or treated with Pg/E2 with or without 1 µM Lactacystin (Lac) or RU486. Cells were fractionated and proteins analyzed by immunoblotting, all described in Materials and Methods. The inset in A shows that lactacystin decreases Skp2 protein levels. C., Pg, Cdh1 binding to APC: Cells were treated with Pg/E2, cell lysates immunoprecipitated with anti-Cdh1 followed by immunoblotting with anti-Cdc27, as described in Materials and Methods. D, diagram depicts APC/Cdh1 as the upstream E3 ligase that ubiquitylates Skp2/Cks1 (of the SCF complex), which is the downstream E3 ligase that ubiquitylates p27 to regulate p27 protein levels.