Figure 4.

Quantifying p65/RelA phosphorylation in response to TNF-α stimulation of monolayer grown in 96-well transwells. (A) Organoid-derived monolayers grown in 96-well transwells (2 wells per condition) were left untreated or stimulated with 40 ng/mL TNF-α on both apical and basolateral surfaces. At indicated time points, cells were lysed and levels of phosphorylated p65 was quantified using an MSD assay. (B) A dose-series of TNF-α was added to both apical and basolateral surfaces of organoid-derived monolayers grown in 96-well transwells (4 wells per condition). At 15 min post stimulation cells were lysed and phospho-p65 was quantified. (C) Organoid-derived monolayers grown in 96-well transwells (3 wells per condition) were incubated with either an anti-TNF-α antibody or an isotype-matched control for 1 h. TNF-α (40 ng/mL) was added to all samples on both apical and basolateral surfaces and cells were harvested by lysis after 15 min of stimulation, followed by phospho-p65 quantification.