Fig. 1. The MAP3K MEKK2 contributes to the aberrant ERK activation occurring with NF1 loss.

a ERK phosphorylation levels were assessed by immunoblotting in primary COBs isolated from Mekk2^+/+ and Mekk2^−/− mice. Cell lysates were immunoblotted with the indicated antibodies. b Primary Mekk2^+/+ and Mekk2^−/− COBs were transfected with the OSE1-Luc (ATF4) reporter (n = 3 biologically independent samples). Mean ± s.e.m., unpaired, two-tailed Student’s t test: **P < 0.01. c Purified unactivated GST-MEK1 or His-MEK2 was incubated with purified GST-MEKK2 and an in vitro kinase assay was conducted. Two types of recombinant GST-tagged MEKK2 as indicated with a dagger and double dagger were used as described in the Methods section. GST-MEK5 was used as a positive control. d Representative blot from three independent experiments. Primary Nf1^fl/fl COBs infected with either vector (Vec) or Cre lentivirus, were cultured for 7 days under differentiation conditions. Levels of NF1, phospho-ERK1/2, and ERK1/2 were analyzed by immunoblotting. e Representative images of immunostaining for p-ERK (red) in femurs from 16 weeks old Nf1^fl/fl and Nf1^fl/fl;Dmp1-Cre mice. White arrows indicate p-ERK positive osteoblasts. Nuclei are counterstained with DAPI (blue) and scale bar indicates 100 µm. Three independent fields were examined per mouse (n = 3 mice per group). f human MSCs (hMSCs) were infected with shRNA lentiviruses expressing control (shCtrl) or NF1 (shNF1) targeting shRNAs and stimulated with FGF2 (25 ng/ml) for the indicated times, then MEKK2 phosphorylation was analyzed by phos-tag electrophoresis. g MEKK2 phosphorylation was analyzed by either phos-tag electrophoresis or immunoblotting with p-MEKK2 antibody in primary Nf1^fl/fl osteoblasts infected with either vec or Cre lentivirus cultured for 14 days under differentiation conditions. All blotting was confirmed by at least three independent repeats. h Immunohistochemistry for p-MEKK2 was performed in femurs from 16-week-old Nf1^fl/fl and Nf1^fl/fl;Dmp1-Cre mice. Scale bar denotes 100 µm. Three independent fields were examined per mouse (n = 3 mice per group). i hMSCs were infected with the indicated shRNA lentiviruses and then stimulated with FGF2 for the indicated times before immunoblotting, and ERK1/2 activation was analyzed by immunoblotting. Except where otherwise indicated, all data shown are representative of at least two independent experiments. All unprocessed blots are provided in Supplementary Fig. 5. Source data are provided as a Source Data file.