Fig. 5: Lkb1-dependent Ptbp1 phosphorylation promotes Scf exon 6 retention to induce the formation of soluble stem cell factor (sScf) and drives DC differentiation.

a Reverse transcription-polymerase chain reaction (RT-PCR) analysis of soluble Scf (including exon 6) to membrane-bound Scf (lacks exon 6) in control or target-gene-silenced primary mouse bone marrow ECs. *P = 0.02 versus Control si by nonparametric Mann–Whitney U test (two-sided). b RNA-binding protein was pull down using oligo-dT beads in indicated conditions and subjected to western blotting of Ptbp1 binding. *P = 0.02 versus vehicle by nonparametric Mann–Whitney U test (two-sided). c RT-PCR analysis of soluble Scf/membrane-bound Scf ratio in vehicle or peptide nucleic acid (PNA)-treated bone marrow ECs. *P = 0.03 versus vehicle by nonparametric Mann–Whitney U test (two-sided). d RNA-binding protein (RBP) was pulled down using oligo-dT beads in WT (vehicle-treated) or PNA competition assay and subjected to western blotting of Ptbp1 binding. FT, flow-through; rb-Ptbp1, RNA binding Ptbp1; WCL, whole-cell lysate. *P = 0.004 versus Control si by nonparametric Mann–Whitney U test (two-sided). e Proteins in in vitro kinase assay reaction mixture were separated on a Phos-tag^TM gel and analyzed by western blotting with the anti-Ptbp1 antibody. # marked the phosphorylated band. Blots are representative of three independent experiments. f Endothelial cells (ECs) were transfected with His-Ptbp1^wt and mutant plasmid and whole-cell lysates were analyzed by western blotting for anti-His after separation in Phos-tag^TM gel. # marked the phosphorylated band. Blots are representative of three independent experiments. g PCR analysis was carried out to detect soluble Scf/membrane-bound Scf ratio in BAECs transfected with Ptbp1^wt and mutant plasmid. *P = 0.003; ^†P = 0.007 by nonparametric Mann–Whitney U test (two-sided). h Flow cytometry analysis of bone marrow cells isolated from WT or Stk11^ec−/− mice treated with either AAV-acGFP or AAV-Ptbp1^T138E and stained for Lin, c-Kit, CD115, CX3CR1, and Flt3. Bar graph summarizes the ratio of MDP/CDP in bone marrow (12-weeks-old, mixed-gender, n = 5–6 each group). *P < 0.001 versus WT and ^†P < 0.001 versus Stk11^ec−/− by nonparametric Mann–Whitney U test (two-sided). i Flow cytometry analysis of spleen/lymph node cells isolated from WT or Stk11^ec−/− mice treated with either AAV-acGFP or AAV-Ptbp1^T138E and stained for CD11c and MHC II or CD11c and B220. Bar graph summarizes the frequency of cDCs and pDCs in spleen, and lymph node (12-weeks-old, mixed-gender, n = 5–6 each group). *P < 0.001 versus WT and ^†P < 0.001 versus Stk11^ec−/− by nonparametric Mann–Whitney U test (two-sided).