Flot-1 and CAP compete for the binding to FRS2.

<p>CAP-GST was immobilized to sepharose and incubated with HeLa cell lysates in the presence of increasing amounts (1–5 µg) of purified FRS2-His. The binding of endogenous flot-1 from the lysates was analyzed by Western blot (upper blot). Middle panel shows the blot for FRS2-His and the lowermost one a ponceau staining of the GST proteins.</p

FRS2 directly interacts with Cbl-associated protein.

<p>(A) Yeast two-hybrid analysis of the interaction between FRS2 and CAP domains. (B) Structure of the CAP-GST constructs used. (C) and (D) Interaction of purified FRS2-His and CAP-GST proteins. CAP-GST fusion proteins were immobilized on sepharose and tested for the binding of purified FRS2-His. Upper blot shows the bound FRS2-His (anti-His antibody), lower blot the ponceau staining of the GST proteins. 1 µg of FRS2-His was used as a positive control. (E) Quantification of the binding of FRS2 to various CAP domains. A binding of FRS2 significantly higher than background was seen with the full-length CAP, delta-SoHo and the third SH3 domain. (F) Endogenous FRS2 was immunoprecipitated from Hep3B cells, and the binding of endogenous CAP was tested. Please note that several isoforms of CAP are present in Hep3B cells, of which only one appears to bind FRS2.</p

Endogenous flot-1 and flot-2 colocalize with FRS2 in Hep3B cells.

<p>Cells were grown in a medium containing FCS and stained with antibodies against endogenous FRS2 (green) and flot-1 or flot-2 (red). Scale bars 10 µm.</p

Overexpression of FRS2 does not compensate for the signaling defects in flot-1 knockdown cells.

<p>FGF receptor and flot-1 compete for the binding to FRS2. (A) Flot-1 was knocked down in HeLa cells by means of siRNAs and the cells were transfected with FRS2-CFP. Starved cells were stimulated with FGF for 5 min, and the activation of Akt (uppermost blot) and ERK2 (3^rd blot) was measured with phospho-specific antibodies. The third blot from the bottom shows the analysis of the transfection efficiency of FRS2-CFP and of the 2^nd one the knockdown efficiency of flot-1. Lowermost blot (GAPDH) shows equal protein loading. (B) Purified FRS2-GST was immobilized on sepharose and incubated with lysates of HeLa cells transfected with increasing amounts of FGFR-myc (0.5 to 2 µg). The binding of endogenous flot-1 from these lysates was tested (upper blot). (C) Quantification of the flot-1 bound to FRS2. In the presence of increasing amounts of FGFR, the binding is significantly reduced. (D) Expression of FGFR was verified by Western blot.</p

Flotillin-1 is required for the recruitment of FRS2 into light membranes in pervanadate treated cells.

<p>Hep3B cells (control: upper panels, flot-1 knockdown: lower panels) were starved overnight and then stimulated with pervanadate. Detergent resistant light membranes were prepared using density gradient centrifugation and found in fractions 1–3 of the gradient. The localization of FRS2, flot-1 and CAP was analyzed. Western blots for transferrin receptor (TfnR), GAPDH and GM1-bound cholera toxin subunit B (CTX-B) were used to control the gradient.</p

Increased Tyr phosphorylation and solubility of FRS2 in flotillin knockdown cells.

<p>(A) FRS2 was immunoprecipitated from serum grown Hep3B cells. The Tyr phosphorylation of FRS2 was measured by means of phospho-Tyr antibodies and found to be increased both in flot-1 and flot-2 knockdown cells. (B) Densitometric quantification of FRS2 phosphorylation with SD (5 independent experiments). F1-KD cells display a significantly increased P-Tyr of FRS2. (C) Hep3B cells were grown under serum, fixed and stained with antibodies against FRS2 (left column) and flotillins (middle). In control cells, FRS2 was localized at the plasma membrane and within the cytosol, whereas in flot-1 or flot-2 knockdown cells, a cytosolic staining was evident. In addition, especially in flot-2 knockdown, some nuclear staining was observed. Right column: overlay with DAPI staining. Scale bars 10 µm.</p

Increased levels of the neutrophil attractant chemokines KC and MIP-2 in WT male mouse sera upon MCMV infection.

<p>WT or TLR9^−/− male and female mice were infected i.p. with 1×10⁵ PFU MCMV and sera were collected 36 h after infection. Upon MCMV infection, WT male mice produced statistically significant higher levels of KC and MIP-2 than WT female or TLR9^−/− male and female mice, while the MCP-1 levels were higher in WT mice compared to TLR9^−/− mice. KC, MIP-2 and MCP-1 protein levels were measured by CBA flex or ELISA. Data are representative of four independent experiments. *p<0.05, **p<0.01 and ***p<0.001.</p

Frequency of splenic B, T, NK, and NKT cell subsets in untreated or MCMV-infected WT or TLR9^−/− male and female mice.

<p>WT and TLR9^−/− male and female mice were left uninfected or infected i.p. with 1,2×10⁵ PFU of MCMV. After, 36 h spleens were harvested, total splenocytes were isolated, stained for B220, CD19, CD3, CD4, CD8, or NK1.1 and analyzed by flow cytometry. The frequency of B cells (CD19⁺B220⁺), CD4 (CD3⁺CD4⁺) and CD8 (CD3⁺CD8⁺) T cells, and NK (B220⁻NK1.1⁺CD3⁻), and NKT (B220⁻NK1.1⁺CD3⁻) cells was determined after gating among live cells. All values denote percentages (mean ± SD) of three mice per group, and are representative of two or three independent experiments.</p

Increased number of neutrophils in the splenic white pulp of MCMV-infected WT male mice.

<p>WT or TLR9^−/− male and female mice were left untreated of infected with 1×10⁵ PFU of MCMV and 4 days later spleens were collected and processed for immunofluorescent analysis. (A) Murine spleen sections were incubated with antibodies specific to neutrophils (7/4, blue or red), B cells (B220, red), marginal metalophilic macrophages (MOMA-1, green) and reticular fibroblasts (ER-TR7, green). MCMV infection led to (A) a dramatic increase in the number of neutrophils that were present in the splenic red pulp and disappearance of the MZ metallophilic macrophages and (B) an increase in the number of neutrophils in the splenic white pulp areas compared to uninfected mice. These phenomena were more prominent in WT male than in WT female or TLR9^−/− male and female mice. (B) Number of neutrophils per white pulp area were counted on slides stained in A. *** p<0.001. Data are representative of 9 mice per group.</p

Increased liver inflammation and steatosis in MCMV-infected female mice.

<p>(A) Livers were harvested and tissue sections were prepared from uninfected (A, C, E and G) or day 3 MCMV-infected male (B and F) and female (D and H) mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045171#s4" target="_blank">Materials and Methods</a>. Hematoxylin and eosin stained liver sections from uninfected (A and C) or MCMV infected (B and D) mice revealed inflammatory and necrotic foci in MCMV-infected mice. Female infected mice had increased inflammation and necrosis (more numerous and bigger foci) compared to male mice. Arrows identify inflammatory and necrotic foci, arrowhead shows an hepatocyte with an enlarged nucleous (karyomegaly) which contains large eosinophilic intranuclear inclusion and pheripheralized chromatin, asterisks indicate intracytoplasmic clear vacuoles. Accumulation of lipids (red stained lipid droplets) was detected by Oil red O staining (E–H) and was significantly more obvious in MCMV-infected female (H) versus male (F) liver sections, while in uninfected (E and G) mice was absent. Magnifications: A–D ×40 and E–H ×20. Data are representative of 2 independent experiments and with 3 mice per group.</p