Skip to main content
. 2012 Sep;82(3):488–499. doi: 10.1124/mol.112.078295

Fig. 5.

Fig. 5.

A, MRP1 and CK2α proteins interact physically. Protein lysates prepared from WT and MRP1 cells were subjected to CoIP with an antibody against CK2α (goat CK2α; lanes 2 and 3) or an antibody against MRP1 (mouse QCRL-1; lanes 5 and 6). Protein A/G PLUS Agarose was added to precipitate the immunocomplex and proteins were analyzed by immunoblotting (IB). Small aliquot of MRP1 lysate was loaded as a positive control for immunoblotting (lanes 1 and 4). Reactions were carried-out in triplicate and blots shown here are representative of the series. B, phosphorylation of MRP1-Thr249 consensus site by recombinant CK2α is dependent on Thr249. The ability of recombinant human CK2α to phosphorylate MRP1-Thr249 consensus site was assessed by in vitro kinase assay as described under Materials and Methods. Synthetic peptides biotin-Ahx-RRRADDSDDDDDK (Control), biotin-Ahx- LNKEDTSEQVV (MRP1244–255), and biotin-Ahx- LNKEDASEQVV (MRP1244- 254T249A) were incubated in the presence of recombinant human CK2α and 32P-labeled ATP. All experiments were normalized to a zero time point and performed in triplicate, and results are reported as nanomoles of phosphorylated peptide formed per 5 min of reaction per milligram of recombinant protein. Statistical analysis was performed using Student's t test (control peptide served as a positive control to test for activity of the recombinant CK2α protein and therefore was not taken into consideration for statistical analysis). C, MRP1-Thr249-P antibody pulls down MRP1 protein from MRP1 cells but not from MRP1-T249A line. Custom-made rabbit antibody against synthetic peptide corresponding to MRP1-Thr249-P consensus site [WSLNKEDT(p)SEQVVP] was used to immunoprecipitate (IP) MRP1 protein from membrane fractions prepared from WT, MRP1, and MRP1-T249A cells, followed by IB with MRP1 antibody (QCRL-1). D, test of linearity carried out by immunoprecipitation with fixed amount of MRP1-Thr249-P antibody on increasing amount of MRP1 membrane fraction. E, knockdown of CK2α results in decreased MRP1 phosphorylation at Thr249. Membrane fractions prepared from MRP1 and MRP1 CK2α(−) cells were subjected to immunoprecipitation with rabbit MRP1-Thr249-P antibody (top). Relative phosphorylation (phosphorylated MRP1/ total MRP1) was determined by densitometric analysis with use of Adobe Photoshop CS4.