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. Author manuscript; available in PMC: 2024 Feb 5.
Published in final edited form as: Biomed Pharmacother. 2023 Oct 20;168:115763. doi: 10.1016/j.biopha.2023.115763

Fig. 2. Exogenous H2O2 and β-receptor agonism induces β2AR Cys-S-sulfination in human SAEC.

Fig. 2.

(A) The ability of acute agonism of β2AR to induce cysteine-S-sulfination was assessed via the clickable S-sulfinic acid-selective probe DiaAlk. SAEC or A-SAEC (4 ×105 cells/well) were seeded in 6-well plates and agonized with (A) ISO (10 μM) for 5–60 min (n = 3) or (B) salbutamol (albuterol; SAL) (10 μM) for 15 or 60 min (n = 3). β2AR cysteine-S-sulfination was assessed by β2AR immunoprecipitation and selective labeling with 1 mM DiaAlk, followed by conjugated with biotin utilizing click chemistry, and detection via streptavidin-HRP, as discussed in the materials and methods. Both ISO (A) and SAL (B) significantly increased β2AR hyperoxidation to cysteine-S-sulfinic acids (upper panels). The amount of β2AR (middle panels) within each reaction is shown as an input control and β-actin (lower panels) is utilized to denote equal protein input and loading. (C) The effects of chronic ISO agonism or H2O2 were also assessed and cells were treated every 12 h with for seven days with either ISO (10 μM) or H2O2 (10 μM). The media was replenished every 2–3 days to prevent nutrient depletion-mediated starvation. Both SAEC and A-SAEC exhibit significantly elevated DiaAlk-biotin labeled β2AR (upper panels), indicative of cysteine-S-sulfination, following the seven-day treatment paradigm with both ISO and H2O2. The amount of β2AR (middle panels) within each reaction is shown as an input control and β-actin (lower panels) is utilized to denote equal protein input and loading.