Alexafluor 568 conjugate

Animals were sacrificed 8 weeks after the microbeads injections. Eyes (n = 24) were dissected in 0.1 M PBS and fixed in 4% paraformaldehyde dissolved in 0.1 M PB for 2 h at room temperature followed by washing 0.1 M PBS for one hour. After the washing steps, we removed the retina from the eyecup and made four small cuts. To stain the retinal vasculature, we placed the retinas in a well plate and added 500 µL of fluoresceinated isolectin solution (Isolectin GS-IB4 from Grifonia simplicifolia, Alexa Fluor568 conjugate; Thermo Fischer Scientific, Waltham, MA, USA). After an overnight rocking incubation in lectin solution at room temperature, retinas were rinsed 6 times in PBS. The labeled, isolated retinas were placed and unfolded on a glass slide. To avoid later bleaching of the fluorophores, we mounted the slides with Fluoroshield (Sigma-Aldrich, Budapest, Hungary) mounting medium. Images were made of the retinal whole-mounts with a Nikon Eclipse 80i epifluorescence microscope.

HEK^ΔSia cells were seeded in 6-well plates (1.5E + 05 cells/well) and the next day cotransfected with a pEGFP reporter plasmid and different amounts and ratios of sialyltransferases (ST6Gal1 and/or ST3Gal4). After 48 h transfected cells were released using Cell Dissociation Buffer (Gibco), washed once in phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde (PFA) in PBS. Cells firstly were incubated with biotinylated-lectins (MAL I, Vector Labs; SNA, Vector labs) for 1 hr and then complexed with Streptavidin for 1 hr, (Alexa Fluor^™ 568 conjugate (1 mg/mL),Thermo Fisher). Cells were analyzed on BC Cytoflex LX (Beckman) using CytExpert fot CtytoFLEX Acquisition and Analysis software. The gating methods are based on standard protocols^{67 (link)}. Representative flow cytometry gating strategy for lectin staining cells are shown in Supplementary Fig. 7.

Whole lungs were perfused with 10% neutral-buffered formalin (NBF) in situ. Tissue was then fixed overnight in 10% NBF, embedded in paraffin and sectioned. Lung sections were treated with a standard antigen retrieval and immunofluorescence staining protocol: DAPI (Life Technologies), anti-myeloperoxidase (anti-MPO) (R&D Systems), anti-Streptococcus pneumoniae type 2 rabbit polyclonal serum (anti-Strep, Abcam), donkey anti-goat IgG (H+L) secondary antibody Alexa Fluor 568 conjugate (Life Technologies) and donkey anti-rabbit IgG (H+L) secondary antibody Alexa Fluor 488 conjugate (Life Technologies); or DAPI, anti-MPO, anti-citrullinated histone 3 (anti-citH3, citrulline R2 + R8 + R17) (Abcam) and the same secondary antibody as above. Stained tissues were mounted and examined with confocal microscopy. Image analysis was performed using ImageJ 1.64.

Uptake assays were used to assess the effect of Dynasore treatment, α-adaptin knock-down and caveolin1 knock-down on endocytosis. 24 h prior to the assay cells were plated onto sterilised glass cover slips. For assays with Dynasore each well was rinsed with serum free media (SFM) before incubation with SFM containing 80 μM Dynasore (Sigma), or the equivalent volume of dimethyl sulphoxide (DMSO), for 30 min. A 10 min incubation with just SFM was used for assays utilising siRNA transfected cells. After this initial incubation cells were then further incubated for 5 min with either transferrin (Alexa Fluor 568 conjugate, 10 µg/mL, Life Technologies) or cholera toxin B subunit (CTxB, Alexa Fluor 555 conjugate, 1 µg/mL, Life Technologies) diluted in SFM. After incubation cells incubated with transferrin were washed with acid PBS twice followed by a PBS wash; cells incubated with CTxB were washed twice with SFM followed by a PBS wash. Cover slips were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) for 5 min. Cover slips were fixed to slides using ProLong Gold (Life Technologies) and imaged using a Nikon TE300 Inverted Epi-fluorescence microscope with a 60× oil objective lens. Random fields of view from each slide were recorded and the light intensity of 20 random cells per slide was analysed.

2 × 10⁵ HEK293T cells were grown on polyK coated 35 mm plates and transfected with 500 μg of either pDNA3.1-Flag, pCDNA3.1-Flag-DcpS pCDNA3.1-Flag-ΔNES or pCDNA3.1-Flag-ΔNLS. 24 h after transfection, cells were fixed with PBS-PFA 4% (w/v). After washing with PBS, permeabilization was performed using PBS- triton X-100 0.5% (v/v) for 5 min, and then washed with PBS. After blocking in PBS-BSA 5% (w/v) for 20 min, anti-Flag M2 (Sigma; dilution 1:500 in PBS-BSA 5%) was incubated for 60 min, and then washed three times with PBS-tween 0.2% (v/v). Secondary Goat anti-Mouse IgG secondary antibody, Alexa Fluor^® 568 conjugate (Life Technologies. dilution 1:250 in PBS-BSA 5%) was incubated for 60 min, and after three washes, cells were mounted with 1 ug/ml DAPI staining.