Fig 2: ERK3 does not phosphorylate SRC-3. (A) MK5, but not ERK3, phosphorylates SRC-3-S857 in vitro. For in vitro kinase assay, either 300 ng of active recombinant ERK3 protein (83.5 kDa) or 50 ng active recombinant MK5 (54 kDa) or both was incubated with 2 µg GST or GST-CID-SRC-3 WT or GST-CID-SRC-3 S857A in kinase buffer and 1 µCi [?32P]-ATP. The reaction was carried out at 30 °C for 15 min. Proteins were resolved by SDS-PAGE gel and visualized by autoradiography. (B) In vitro kinase assay was performed by incubating 2 µg GST or wild type (WT) or mutant (S857A) GST-CID-SRC-3 fusion proteins with and without 50 ng active MK5 in the kinase buffer for 15 min. Serine 857 phosphorylation and total amount of GST-CID-SRC-3 WT and GST-CID-SRC-3 S857A fusion proteins were detected by Western-blotting using anti-P-S857-SRC-3 and anti-GST antibodies, respectively. The full-length blots are presented in supplementary figure S4. (C) MK5 phosphorylated GST-CID-SRC-3 fusion protein (2 µg) was diluted 2, 4, 8, 16 and 32 times before separation on SDS-PAGE followed by Western-blotting. The membrane was then probed with anti-GST and anti-P-S857-SRC-3 antibodies. The full-length blots are presented in supplementary Figure S5. (D) H1299 wild type cells were seeded in 6-well plates and left overnight followed by transfection with 1 µg vector encoding either SRC-3 wild type-FLAG (SRC-3 WT-FLAG) or SRC-3 S857A-FLAG (SRC-3 S857A-FLAG). After 48 h of transfection, the cells were lysed. FLAG-tagged SRC-3 and level of serine 857 phosphorylation of SRC-3 in the lysate was detected by Western-blotting with anti-FLAG and anti-P-S857-SRC-3 antibodies, respectively. The full-length blots are presented in supplementary figure S6. (E) Endogenous SRC-3 protein was immunoprecipitated from H1299 cells. After the last wash step, half of the precipitate was treated for 30 min with 400U lambda phosphatase. Western-blot was performed with anti-SRC-3 and anti-P-S857-SRC-3 antibodies. The full-length blots are presented in supplementary Figure S7.