Fig. 7. NLK phosphorylation of c-Myb results in ubiquitination and proteasome degradation.

a CD34⁺ CB HSPCs transduced with shRNA against control (shLuc) or RPS19 (shRPS19) were differentiated for 10 days and sorted into CD71⁺ and CD71^– fractions. Three independent experiments were pooled and populations were probed by Western blot analysis examining phospho-Ser863-Raptor and total Raptor. This experiment was performed in conjunction with Fig. 3c. b Cord blood CD34+ progenitors were transduced with shRNA against luciferase (shLuc) or RPS19 (shRPS19) and siRNA against NLK (siNLK) or a non-targeting (NT) sequence and differentiated for 8 days. Cells were fixed, permeablized and incubated with Cy3-labeled antibody against Raptor (pseudo-colored red) and lysosomes were visualized by incubation with FITC-labeled antibody recognizing LAMP1 (pseudo-colored green). Areas of co-localization (merge) are indicated in yellow and yellow dotted boxes are magnified in inserts to the far right. Scale bar, 15 µm c Fetal liver CD34⁺ progenitors were transduced with shRNA against luciferase (shLuc) or RPS19 (shRPS19) in conjunction with siRNA against NLK (siNLK) or a non-targeting sequence (NT). Cells were differentiated for 5 days and split into two aliquots. One aliquot was lysed and probed for c-Myb protein expression by Western blot, while the second aliquot was subjected to qRT-PCR to examine c-Myb mRNA expression. d Kp53A1 cells were treated with vehicle alone, lactacystin or chloroquine for 30 min, prior to switching cells from 37 to 32 °C for the indicated times. Cells were lysed, normalized for protein and split into two. C-Myb was immunoprecipitated from one sample before Western blot analysis for phosphorylated serine, while the other sample was subjected to Western blot for c-Myb and GAPDH. Bars represent means ± SD with individual data points overlaid. n = 3 independent experiments performed in triplicate. Also see Supplementary Fig. 7. Source data are provided as a Source Data file.