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. 2020 Nov 11;11:5704. doi: 10.1038/s41467-020-19555-6

Fig. 3. Pharmacologic inhibition of MEKK2 prohibits ERK activation.

Fig. 3

a Primary COBs were immortalized through infection with a retrovirus expressing SV40 large T-Antigen. COBs were pre-treated with the indicated inhibitors (1 µM, 30 min), then stimulated with or without FGF2 (25 ng/ml, 20 min) 30 min later. MEKK2 phosphorylation was analyzed by phos-tag electrophoresis. b MEKK2 phosphorylation was analyzed by either phos-tag electrophoresis or immunoblotting with anti-p-MEKK2 in primary Nf1fl/fl osteoblasts infected with either vec or Cre lentivirus with the indicated inhibitors and FGF2 stimulation. c hMSCs were treated with the indicated inhibitors, then MEKK2 phosphorylation was examined by phos-tag electrophoresis. d WT immortalized COBs were lysed and immunoblotted for the indicated antibodies after inhibitor treatment. e Activation of ERK was detected in Saos-2 cells after incubation with the indicated inhibitors and stimulation with or without FGF2. f Purified unactivated GST-MEK1 was incubated with purified GST-MEKK2 and the indicated doses of ponatinib, and kinase activity of MEKK2 was analyzed by an in vitro kinase assay. g Expression levels of osteoblast genes in primary Nf1fl/fl osteoblasts infected with either vec or Cre lentivirus. After infection, these cells are treated with the indicated inhibitors or a Mekk2-targeting shRNA and cultured for 14 days (n = 4 biologically independent samples). mean ± s.d. h Saos-2 cells were treated with DMSO, ponatinib (1 µM), BRITE-0600690 (BRITE-690, inactive compound), and BRITE-0600719 (BRITE-719, active compound) for 1 h with or without FGF2 stimulation. Phosphorylation levels of ERK1/2 were assessed by immunoblotting. All data shown are representative of either two or three total independent experiments. All unprocessed blots are provided in Supplementary Fig. 5. Source data are provided as a Source Data file.