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Biochemical Characterization of TIPE2 Signaling

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Assays were performed as we previously described^{10 (link),18 (link),43 (link)}. The purity of BMDMs populations was greater than 95% as determined by flow cytometry after staining cells with anti-CD11b-FITC (eBioscience) and anti-F4/80-APC (eBioscience), and the viability was greater than 99% as determined by trypan blue staining. Antibodies to the following antigens were used for immunoblotting and co-immunoprecipitation: Rac1 (EMD Millipore), Rac1/2/3, RhoGDI, AKT, p-AKT (T308), p-AKT (S473), p-GSK-3β (S9), cofilin, p-cofilin (S3), p-PAK1/2 (S144/S141), p-LIMK1 (Thr508)/LIMK2 (Thr505), ERK1/2, p-ERK1/2 (T202/Y204), p-p38 (T180/Y182), mTOR, GAPDH, integrin-β1 (Cell Signaling Technology), TIPE2 (Proteintech), Flag, actin (Sigma-Aldrich,), HA (Cell Signaling Technology). Control IgG (Santa Cruz Biotechnology) anti-rabbit IgG-HRP, and anti-mouse IgG-HRP (GE Healthcare Life Sciences) were also used.

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Fayngerts S.A., Wang Z., Zamani A., Sun H., Boggs A.E., Porturas T.P., Xie W., Lin M., Cathopoulis T., Goldsmith J.R., Vourekas A, & Chen Y.H. (2017). Directing leukocyte polarization and migration by the phosphoinositide transfer protein TIPE2. Nature immunology, 18(12), 1353-1360.

Publication 2017

anti-CD11b-FITC anti-F4/80-APC p-AKT p-GSK-3β cofilin p-cofilin p-PAK1/2 p-LIMK1 p-ERK1/2 GAPDH integrin-β1 TIPE2 Control IgG anti-rabbit IgG-HRP anti-mouse IgG-HRP

Actin Anti igg Antibodies Antigens Assays Cd11b Cells Co immunoprecipitation Cofilin Erk1 Fitc Flow cytometry Gapdh Integrin Limk1 Limk2 Mouse Mtor Pak1 Populations Rabbit Rhogdi Trypan blue

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