Fig 2: Ala255 of LYN is required for minocycline binding. (A-B) Cartoon representation (A) and electrostatic surface representation (B) of the docking model of minocycline binding to H. sapiens LYN. (C) The possible binding sites of LYN to minocycline were determined by docking analysis. (D) Following transfection of LYNWT or LYN mutant plasmids in 293T cells, the Flag-LYNWT and LYN mutant proteins were affinity-purified with Epoxy-actived µSphere immobilized with minocycline, and then immunoblot analysis with anti-Flag antibody. (E) Following transfection of LYN mutant plasmids in 293T cells with or without minocycline treatment, western blot analysis was performed with antibodies against p-LYN and LYN.

Fig 3: Effects of SG-SP1 on activation of NF-?B, and signaling proteins in mast cells. RBL-2H3 were sensitized with anti-DNP IgE. After incubation overnight, the cells were pretreated with or without SG-SP1 and GA for 1 h and then challenged with DNP-HSA. Activation of NF-?B and signaling proteins were assayed by Western blot (N-: nuclear, p-: phosphorylated). Lamin B1, ß-actin, and total form of Lyn, Fyn, Syk, and Akt were used as loading controls. The band intensity was quantified and presented as the means ± SEM of three independent experiments. *, **, and *** indicate statistically significant differences from the DNP-HSA-challenged group at a value of p < 0.05, 0.01, and 0.001, respectively.

Fig 4: Predicted ubiquitination site in the LynA insert.Alignment of the amino acid sequence of the two isoforms of mouse Lyn reveals lysine and tyrosine residues unique to LynA. Lysine K40 in LynA only is predicted to be a site of ubiquitination.DOI: http://dx.doi.org/10.7554/eLife.09183.022

Fig 5: Laddering of activated Lyn in whole-cell lysate.We treated CskAS BMDMs with 3-IB-PP1 (10 µM) or medium alone and observed laddering in activated SFK and total LynA, but not total Hck or inactive SFK immunoblots.DOI: http://dx.doi.org/10.7554/eLife.09183.021