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. 2014 May 27;2:22. doi: 10.3389/fcell.2014.00022

Figure 1.

Figure 1

HeLa cells predominantly express the GRAF1c isoform, which interacts with the hub-protein, MICAL-L1. (A) The domain architecture of the three GRAF1 isoforms. (B) GRAF1 isoforms (a, b, and c) were amplified from either human brain or HeLa cDNA libraries with two primers as indicated in (A). (C) Cells were either left in culture, or transfected with either HA-GRAF1b, or HA-GRAF1c for 16 h. In the left panel, the cells were subsequently mock-treated or treated with GRAF1-siRNA for 72 h before being lysed on ice. Lysates were subjected to 8% SDS-PAGE, followed by immunoblotting with anti-GRAF1 and anti-Actin antibodies (loading control). (D) Cells were either transfected with HA-MAPK as a negative control or HA-GRAF1c. After 48 h, cells were lysed for 1 h in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 and protease inhibitors. Cell lysates were incubated with mouse anti-MICAL-L1 antibody overnight. Protein L beads were added to the mixture of cell lysate and MICAL-L1 antibody for 3 h at 4°C. Beads were washed in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Triton. Proteins were eluted by adding SDS loading buffer. Samples were subjected to 8% SDS-PAGE, followed by blotting with anti-HA and anti-MICAL-L1 antibodies.