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. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: J Cell Physiol. 2016 Mar;231(3):630–640. doi: 10.1002/jcp.25108

Figure 4. A non-canonical reversed docking motif in MITF is essential for MITF-p38MAPK interactions.

Figure 4

A. MITF amino acid sequence near the S307 p38 phosphorylation site, aligned with the D type docking motif. The residues where point mutations were introduced for the experiments described in B are shaded grey. Amino acids indicated as φ are hydrophobic, θ indicates a charged residue, and X can be any amino acid.

B. Lysates from COS-7 cells overexpressing His-p38 and FLAG-MITF or FLAG-MITF with various single or double point mutations in the putative p38 docking site as indicated were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-His antibody (top panel). Anti-His and anti-FLAG input controls are shown in the bottom two panels.

C. Lysates from COS-7 cells overexpressing MKK6 and wildtype (wt) FLAG-MITF, FLAG-MITF with the S307A point mutation or FLAG-MITF with the p38 docking site ablating double mutation V311A;R313A as indicated were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) with anti-phospho-S307 MITF antibody (top panel), anti-Pp38 antibody (second panel), or anti-FLAG antibody for pulldown control (third panel from top). Anti-Pp38 input control is shown in the bottom panel.

D. ChIP-qPCR analysis of Pp38 binding to the Ctsk promoter in in vitro differentiated osteoclasts from Mitfce/ce mice and Mitfce/+ controls, n = 2. Primary bone marrow derived myeloid cells were treated in vitro with CSF1 and RANKL for 3 days. Data are represented as the mean +/− S.D.