Fig 5. Deletion of St6gal1 in primary macrophages suppresses TNF and LPS signaling.

(A) Mice with floxed St6gal1 were cross-bred with mice expressing Cre recombinase under control of the LysM promoter (LysM/Cre-ST6^fl/fl). Mice with floxed St6gal1, but lacking Cre recombinase (ST6 ^fl/fl), served as the control population. Monocytes were isolated from the bone marrow and exposed to M-CSF for 4–6 days to obtain BMDMs. Knockout of St6gal1 in LysMCre/ST6^fl/fl mice was confirmed by immunoblotting. (B) Control and St6gal1 knockout BMDMs were stained with SNA-FITC and surface sialylation analyzed with flow cytometry. (C) Control and St6gal1 knockout BMDMs were treated with TNF for 2 or 6 hr, and lysates immunoblotted for phospho- and total NFκBp65. (D) Control and St6gal1 knockout BMDMs were treated with LPS for 2 or 6 hr, and lysates immunoblotted for phospho- and total NFκBp65. (E) Control and St6gal1 knockout BMDMs were treated with LPS for 2 or 6 hr, and lysates immunoblotted for phospho- and total STAT1 and STAT3. (F) Control and St6gal1 knockout BMDMs were treated with IFNγ for 2 or 6 hr, and lysates immunoblotted for phospho- and total STAT1 and STAT3. (G) Control and St6gal1 knockout BMDMs were treated with IL-6 for 2 or 6 hr, and lysates immunoblotted for phospho- and total STAT3. (H) Control and St6gal1 knockout BMDMs were treated with GM-CSF for 2 or 6 hr, and lysates were immunoblotted for phospho- and total STAT5.