Figure 4(A) Amino acid sequence alignment of Sox4 GSK3 phospho-motif from mouse, rat, human and chimp. Mouse serine 316 (S316) (highlighted in green) and S315 (highlighted in yellow) predicted by PhosphoSitePlus to be phosphorylated. For S316 mutant, serine 316 mutated to alanine and for S316all mutant all highlighted serines mutated to alanine. Red highlight indicates proline residue following mutated serine. (B) Amino acid sequence alignment of Sox11 GSK3 phospho-motifs from mouse, rat, human and chimp. Mouse S244 and S289 (highlighted in green) predicted by PhosphoSitePlus to be phosphorylated. For S244S289 mutant, serines 244 and 289 mutated to alanine and for S244allS289all mutant all highlighted serines mutated to alanine. Red highlights indicate proline residues following mutated serines. (C) Analysis of Flag-tagged Sox4 wildtype (WT) protein overexpressed in HEK293T (293T) cells and detected with FLAG antibody. Lysates were treated with or without lambda-phosphatase (λ-Phos). Antibody against phosphorylated β-catenin (pβCAT) was used to validate λ-Phos treatment. Purple arrowheads indicate Sox4 phosho-shift from 70kDa to 65kDa. ERK blot shows similar loading of samples. (D) Analysis of Flag-tagged Sox11 WT protein overexpressed in 293T cells and detected with FLAG antibody. Lysates treated with or without λ-Phos. Upper smear (red arrowhead) represents phosphorylated Sox11. Antibody for pβCAT was used to validate λ-Phos treatment. (E) Analysis comparing molecular masses of WT Sox4 to S316 and S316all mutants. ERK blot shows similar loading of samples. (F) Analysis comparing molecular masses of WT Sox11 to S244S289 and S244allS289all mutants. Red arrowheads indicate downward shifts of phosphorylated Sox11. ERK blot shows similar loading of samples. (G) Co-transfection of myc-tagged GSK3β and Flag-Sox4 in 293T cells. BIO treatment (0.5 µM) performed for 1 hour prior to harvest. Lower band (65kDa) represents non-phosphorylated Sox4. High and low exposure (exp) blots shown to better visualize non-phosphorylated Sox4. Numbers above blot are quantification of Sox4 protein levels normalized to GFP and are expressed as fold change vs. controls. The sum of upper and lower bands was quantified (n=8 biological replicates). (H) Myc-GSK3β and S316all mutant co-transfection in 293T cells plus 0.5 µM BIO treatment one hour prior to harvest. Protein levels quantified as in (G) (n=4 biological replicates). (I) Myc-GSK3β and Flag-Sox11 co-transfection in 293T cells. BIO treatment (0.5 µM) performed for 1 hour prior to harvest. Upper band represents phosphorylated Sox11. Numbers above blot are quantification of Sox11 protein levels normalized to GFP and are expressed as fold change vs. controls. The sum of lower and upper bands was quantified (n=14, experiment performed 4x in triplicate and once in duplicate). (J) Myc-GSK3β and S244allS289all mutant co-transfection in 293T cells plus 0.5 µM BIO treatment one hour prior to harvest. Protein levels quantified as in (I) (n=6, experiment performed 2x in triplicate). (K) In vitro kinase assay on immuno-purified Sox4WT and S316all mutant using recombinant GSK3β protein and radiolabelled ATP. Samples were treated with λ-Phos prior to assay and run on Phos-tag gel. Coomasie Blue staining shows equal loading of WT and mutant proteins. (L) In vitro kinase assay of purified Sox11WT and S244allS289all mutant using recombinant GSK3β protein and radiolabelled ATP. Experiment performed as in (K).