Phos tag agarose

Kinase assays were performed in 20 μl reaction mixture containing wit 1 mM cold ATP (Thermo Fisher), 50 mM Tris–HCl pH 7.0, 10 mM MnCl₂, 250 ng of recombinant POSTN and 500 ng of FAM20C-WT-V5/His or -D478A-V5/His protein. The total protein amount was kept constant supplemented by BSA. The mixtures were incubated for 30 min at 30 °C, Phos-tag agarose (Wako USA) was added, further incubated for 1 h at 4 °C, washed with lysis buffer and SDS sample buffer was added. Reaction products were separated by 4–12% SDS-PAGE and detected by Western blotting using anti-Periostin antibody.

The soluble forms of FLAG-tagged FAM20B and FAM20C were purified from the conditioned media of COS-1 cells transfected with the respective FLAG-tagged expression vectors. The reaction mixture for the protein kinase assay (50 µl) contained 500 ng of purified FLAG-tagged FAM20 proteins, 50 mM HEPES (pH 7.4), 10 mM MnCl₂, 1 mM ATP, and an acceptor substrate (0.625 µg of recombinant human C4ST-1, #11396-H08B, Sino Biological, Beijing, China). The mixtures were incubated at 30 °C for 2 h and subjected to phosphate affinity chromatography^{66 (link)} using phos-tag agarose (Fujifilm Wako), according to the manufacturer’s instructions. The presence of phosphorylated C4ST-1 was analyzed by immunoblotting of the resultant phos-tag-bound materials using an anti-C4ST-1 antibody (clone L18).