Skip to main content
. 2016 Jan 1;129(1):191–205. doi: 10.1242/jcs.179911

Fig. 2.

Fig. 2.

PHD1 phosphorylation at S130 is regulated by CDKs. (A) 300 µg of U2OS GFP or PHD1–GFP cell extracts were subjected to immunoprecipitation (IP) using GFP-trap beads, and precipitated material was analysed by western blotting using the indicated antibodies. (B) 300 µg of U2OS GFP or PHD1–GFP cell extracts were subjected to immunoprecipitation using antibodies towards CDK4, CDK6 and CDK2, and precipitated material was analysed by western blotting for the presence of PHD1. (C) 500 µg of U2OS cell extracts were subjected to immunoprecipitation using an anti-PHD1 antibody crosslinked to Sepharose beads and processed as in A. (D) U2OS PHD1–GFP cells were transfected with control, CDK1 or CDK2 siRNA (siCDK) oligonucleotides alone or in combination for 48 h prior to lysis for western blotting or fixation for FACS analysis. Cell lysates were analysed for the levels of phosphorylated PHD1 at S130 and appropriate controls. The middle graph depicts the mean±s.d. of the quantification of the western blot analysis, representing a minimum of three independent experiments. The right panel depicts the corresponding cell cycle profile of cells treated as mentioned (mean±s.d. of a minimum of three independent experiments). (E) U2OS PHD1–GFP cells were transfected with control or the indicated CDKs siRNAs alone or in combination for a period of 48 h prior to being processed and analysed as in D. (F) U2OS PHD1–GFP cells were transfected with 1 µg of control or CDK4 expression constructs for 48 h prior to lysis and analysed by western blotting for the levels of phosphorylated PHD1 at S130 and appropriate controls. Ev, empty vector control. Graph depicts mean±s.d. of the quantification of the western blot analysis, from a minimum of three independent experiments. (G) CDK2 was immunoprecipitated from cells and used in a kinase assay with 2 µg of recombinant PHD1 and PHD1-S130A protein. Reactions were analysed by western blotting with the indicated antibodies. *P<0.05, **P<0.01 and ***P<0.001 compared to control conditions (Student's t-test). p, phosphorylated form of the protein. See also Fig. S1.