Fig 1. Knockdown of ST6Gal-I expression decreases sustained activation of NFκB by TNF and LPS.

(A) U937 monocytic cells were stably transduced with lentivirus encoding shRNA for ST6Gal-I. Control cells were generated by transduction of an empty vector (EV) lentiviral construct. ST6Gal-I knockdown (ST6-KD) was confirmed by immunoblotting. (B) Surface sialylation levels were measured by staining cells with SNA-FITC, followed by flow cytometry. (C) U937 EV and ST6-KD cells were treated with TNF for 15–30 min (“short-term” timepoints), or 2–6 hr (“long-term” timepoints), and lysates were immunoblotted for phospho-NFκB (p-NFκBp65) or total NFκB (NFκBp65). (D) EV and ST6-KD cells were treated with LPS for short-term or long-term timepoints and lysates were immunoblotted for p-NFκBp65 or total NFκBp65. Dotted lines indicate that blots were spliced to remove irrelevant, intervening lanes (i.e., all lanes shown were on the same blot).