(Right) The lack of inhibition of Siglec\F expression by the PI3K inhibitor

(Right) The lack of inhibition of Siglec\F expression by the PI3K inhibitor. and cell surface expression of Siglec\F was examined by indirect staining. Anti\Siglec\F antibody (R&D Systems) and control rat IgG (Santa Cruz Biotechnology) were used for primary antibody. A representative result of 3 independent experiments is shown. IMM-158-340-s002.tiff (371K) GUID:?83DCC344-037B-40D7-8A0E-060B8D3F1006 Figure S3. IL\3 enhances Siglec\F expression in BMDMs. M\BMDMs were stimulated with IL\3 for 24 h and Siglec\F expression was examined by qRT\PCR. Data are the mean SE of 5 independent experiments. * 005 versus none by the Students 005 versus the control by the Students 005 versus the control by the Students 005 versus the control at the same time point by the Students 005 versus the control by the Students 005 versus the control by the Students (IFN\in macrophages stimulated by Toll\like receptor ligands or group B Streptococcus0111:B4) was purchased from Sigma\Aldrich (St. Louis, MO). Thioglycollate was obtained from Eiken Chemical (Tokyo, Japan). The mitogen\activated protein kinase kinase (MEK) inhibitor PD0325901 and phosphoinositide 3\kinase (PI3K) inhibitor wortmannin were purchased from Wako Chemicals (Osaka Japan) and Cayman Chemical (Ann Arbor, MI), respectively. The STAT5 inhibitor (CAS 285986\31\4) was obtained from Calbiochem (Darmstadt, Germany). The following antibodies were used in the analysis and purification of cells. A phycoerythrin\labeled rat anti\Siglec\F (E50\2440) antibody and an unlabeled rat anti\Siglec\F (238023) antibody were obtained from BD Biosciences (San Jose, CA) and R&D Systems (Minneapolis, MN), respectively. Fluorescein isothiocyanate (FITC) \labeled rat anti\F4/80 (BM8.1) and allophycocyanin (APC) \labeled rat anti\CD11b (M1/70) antibodies were from TONBO Biosciences (San Diego, CA). Fc block (anti\CD16/CD32 (Fc receptor) antibody, 2.4G2, 553141) was from BD Biosciences. Phycoerythrin\labeled control rat immunoglobulin G2a (IgG2a) (RTK2758) was obtained ROBO4 from Biolegend (San Diego, CA). Antibodies were used at 1?g/ml in the flow cytometric analysis. Antibodies towards phospho\p44/42 (Thr202/Tyr204) mitogen\activated protein kinase [phospho\extracellular signal\regulated kinase (pERK), #9101], phospho\Akt (Ser473, #9271), and phospho\STAT5 (Y694, C11C5, LH 846 #9359) were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti\phospho\STAT6 (Tyr641, sc\11762) and anti\I(C21, sc\371) antibodies were from Santa Cruz Biotechnology (Dallas, TX). A rabbit anti\Arg1 (GTX109242) antibody was from GeneTex (Irvine, CA). Rabbit anti\ERK (51068\1\AP) and anti\Akt (10176\2\AP) antibodies were from Proteintech (Rosemont, IL). Rabbit anti\STAT5 (AF2168) and anti\STAT6 (HPA001861) antibodies were obtained from R&D Systems and Sigma\Aldrich, respectively. A mouse anti\chain and GATA118) were lower LH 846 than 02% that of eosinophil freshly isolated from peritoneal cavity (data not shown). In some experiments, macrophages were purified based on the expression of CD11b and F4/80 using FACSJazz (BD Biosciences) as follows. Harvested M\BMDMs were initially incubated with LH 846 Fc block, and stained with APC\labeled anti\CD11b and FITC\labeled anti\F4/80 antibodies. CD11b+?F4/80+ cells were sorted as macrophages (see Supplementary material, Fig. S1a). In order to obtain thioglycollate\elicited peritoneal cavity\derived macrophages, C57BL/6 mice were injected intraperitoneally with 25?ml of 3% thioglycollate. After 72?hr, peritoneal cells were harvested by lavage LH 846 with 067% EDTA/PBS. Cells were incubated with Fc block, and then stained with phycoerythrin\labeled anti\Siglec\F, APC\labeled anti\CD11b, and FITC\labeled anti\F4/80 antibodies. Siglec Flow?CD11b+?F4/80+ cells were sorted as macrophages (see Supplementary material, Fig. S1b). In some experiments, Siglec\Fhigh cells were sorted as eosinophils to compare the expression of Siglec\F. Cell stimulation Macrophages (40??105) were seeded in RPMI\1640 medium supplemented with 10% fetal calf serum, 100?U/ml penicillin, and 100?g/ml streptomycin on 24\well plates and cultured for 2C4?hr to allow attachment. Medium was replaced with fresh LH 846 medium to remove non\adherent cells. Cells were then stimulated in the absence of M\CSF. Unless otherwise stated, 10?ng/ml GM\CSF, 20?ng/ml IL\4, 10?ng/ml LPS, 10?ng/ml IFN\test. A value ?005 was considered to be significant. Results Up\regulation of Siglec\F by GM\CSF in macrophages The expression of CD33\related Siglecs in BMDMs activated by various stimuli was analyzed. Mouse bone marrow cells were cultured with M\CSF to induce M\BMDMs. These cells were confirmed to express iNOS (M1 marker) and Arg1 (M2 marker) mRNA by LPS plus IFN\and IL\4, respectively, as judged by qRT\PCR (data not.