However, the amount of phosphorylated PLC2 and PKC isoforms (PKC, PKC, PKC) were below the limit of detection

However, the amount of phosphorylated PLC2 and PKC isoforms (PKC, PKC, PKC) were below the limit of detection. Dectin-1, is involved in macrophage phagocytosis of for 1 h. Percentages of cells taking up were analyzed by flow cytometey. Mean SD are shown (n = 3). * Q 0.05. NS, not significant [one-way ANOVA with Tukey post-hoc analysis].(TIF) ppat.1004985.s002.tif (159K) GUID:?A9524434-BAF6-4DE5-B6D2-0DDFD97569FA S3 Fig: Viable activates macrophage Syk-JNK-AP-1 pathway and cytokine response through CR3 and Dectin-1. Macrophages from WT, Q 0.05, ** Q 0.01, *** Q 0.001 [one-way ANOVA with Tukey post-hoc test analysis (A)].(TIF) ppat.1004985.s003.tif (949K) GUID:?14316ED0-6D81-4F22-9116-4F8DFFFAA03E S4 Fig: Antibody blockade of Rabbit polyclonal to ALX3 CR3 and Dectin-1 reveals their collaboration in for another 6 h. The concentrations of TNF and IL-6 in culture supernatants were quantified by ELISA. Data shown are the mean SD of relative TNF and IL-6 (n = 5). (B) Macrophages from WT mice were treated with isotype control or blocking antibodies against CR3, Dectin-1, or both for 1 h before stimulation with HK Q 0.001 [2-tailed for 15, 30 and 60 min. Cell lysates were analyzed by Western blotting for activated Syk (A), JNK (B), AP-1 and NF-B (C). The intensity of p-Syk (A), p-JNK (B), p-IB, IB and p-NF-Bp65 at 30 min and p-c-Jun and p-c-Fos at 60 min (C) after stimulation was normalized against the corresponding internal controls. Data shown in the right panel of (A-C) are the mean SD of relative intensity (n = 5). ** Q 0.01, *** Q 0.001 [2-tailed for 30 min. (A) Cell lysates were subjected to sucrose gradient ultracentrifugation. The presence of Syk in each fraction was analyzed by Western blotting. The blot probed with anti-flotillin-1 antibody was used to identify lipid raft fractions. (B) Bar graph shows the relative ratio of Syk to flotillin-1. That in fraction 4 of unstimulated cells was set as 1. Data presented are the mean SD (n = 3).(TIF) ppat.1004985.s008.tif (187K) GUID:?0F89A66A-3845-4412-A4D2-22F3265669D6 S9 Fig: Micro-Western Array screening for signaling protein activation in macrophages stimulated with were screened by Micro-Western Array (MWA) to measure the changes in abundance of indicated proteins. The six samples printed in each well from left to right are macrophages unstimulated (0 min), and stimulated with HK at a yeast-to-cell ratio of 20/1 for 15, 30, 60, 90, and 120 min. The red and green signals represent samples probed with secondary anti-rabbit and anti-mouse antibodies, respectively. The fluorochrome intensities were analyzed by Odyssey analysis software. S1 Table lists the antibodies used for blotting in each well of the 96-well array.(TIF) ppat.1004985.s009.tif (9.7M) GUID:?6EFEAE0E-EEEC-43D6-8973-0E94FC9348FE S10 Fig: CR3 and Dectin-1 are involved in dendritic cell IL-12 response to (MOI = 2) for 6 h. The expression levels of IL-12p35 (A) and IL-12p40 (B) mRNA were analyzed by real-time qPCR. Data shown are the mean SD of relative transcript normalized against GAPDH (n = 3). * Q 0.05, *** Q 0.001 [one-way ANOVA with Tukey post-hoc analysis].(TIF) ppat.1004985.s010.tif (195K) GUID:?54A20140-3EFF-47C7-AAB3-1A8EE5115ACA S11 Fig: Percentage and number of leukocyte populations in spleens from intravenously. Infected mice were killed on day 9 after infection. The percentage (A) and number (B) of CD4+, CD8+, B220+, CD11c+ and Ly6G+ cells in the spleen was analyzed by flow cytometry. Mean SD Dehydrocholic acid are shown (n = 3-4). * Q 0.05, ** Q 0.01, *** Q 0.001 [one-way ANOVA with Duncan post-hoc analysis].(TIF) ppat.1004985.s011.tif (928K) GUID:?0C703DAF-FCF1-4175-9BDE-36206BCD82A5 S12 Fig: Expression of -(1,3)-glucan and -(1,3)-glucan on and C. albicans. (A) strain 505 has -glucan exposed and lacks -glucan expression on the yeast cell wall. Live or HK was stained with anti–glucan or anti–glucan antibody in the presence or absence of laminarin and analyzed by flow cytometry. (B) strain G186A expressing -glucan masks -glucan on the yeast cell wall. Viable wild-type or strain G186A were stained for surface expression of -(1,3)-glucan and -(1,3)-glucan and analyzed by flow cytometry. (C) Heat treatment exposes -glucan on the surface of Dehydrocholic acid strain SC5314 were stained for surface -(1,3)-glucan and analyzed by flow cytometry.(TIF) ppat.1004985.s012.tif (1.2M) GUID:?0369D5D4-9C70-48C9-9BA5-D63516ECB45D S13 Fig: Inhibition of NF-B does not affect infection. WT and intratracheally. Mice were Dehydrocholic acid killed on day 7 post-infection. Leukocytes were.