Anti s100a9 clone d3u8m

Colonic cells were lysed with cell lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors (Cell Signaling Technology). Cell lysates were analyzed by Western blotting according to standard protocol using the indicated antibodies: anti-pSTAT3 (Y705) (clone D3A7; Cell Signaling Technology), anti-STAT3 (clone 79D7; Cell Signaling Technology), anti-pp65 (clone 93H1; Cell Signaling Technology), anti-p65 (clone D14E12; Cell Signaling Technology), anti-S100a8 (clone E4F8V; Cell Signaling Technology), anti-S100a9 (clone D3U8M; Cell Signaling Technology).

20μg of S protein was administered intranasally to WT and Dok3^-/- mice. PBS was used as a negative control. At 24h post-administration, lungs were harvested for flow cytometry analysis or fixed in 4% PFA overnight. For immunohistochemistry, lung sections were stained with anti-S100a8 (clone E4F8V; Cell Signaling Technology), anti-S100a9 (clone D3U8M; Cell Signaling Technology), according to manufacturer’s instructions.

Cell suspensions were surface labelled with fluorochrome-conjugated antibodies for 10 minutes at 4°C in staining buffer (PBS containing 1% BSA). For phosphoprotein staining, cells were fixed and permeabilized using the Phosflow kit (BD) according to manufacturer’s protocol before staining for 1 hour at room temperature. Data were acquired using LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star). The following antibodies were used for flow cytometry analysis: anti-CD45 APC/Cy7 (clone 30-F11; BioLegend), anti-Ly6G PE/biotin (clone 1A8; BD, BioLegend), anti-STAT3 (Y705) PE (clone 4/P-STAT3, BD Biosciences), anti-STAT3 (S727) PE (clone 49/P-STAT3, BD Biosciences), anti-pJAK2 (Y1007,1008) Alexa Fluor 647 (clone E132, abcam), anti-Erk1/2 (pT202/Yp204) PE (612593, BD Biosciences), anti-pIKKα/β (S176/180) PE (14938S, Cell Signaling Technology), anti-p-p38 (T180/Y182) FITC (612594, BD Biosciences), anti-S100a8 (clone E4F8V; Cell Signaling Technology), anti-S100a9 (clone D3U8M; Cell Signaling Technology), anti-pSHP2 (Y580) PE (MA5-28045, Invitrogen), goat anti-rabbit IgG (H+L) secondary antibody FITC (Invitrogen).