FIGURE 2.

TGF-β1 increased LMTK2 abundance at the apical plasma membrane of polarized CFBE41o- and differentiated HBE cells. Representative immunoblots (A) and summary of experiments (B) demonstrating increased LMTK2 levels at the apical membrane after 30 min of TGF-β1 stimulation, and reaching maximum at 1 h. CFBE41o- cells were cultured on Transwell filters to allow polarization and treated with TGF-β1 (15 ng/ml) or vehicle control for 5, 15, 30, 60 or 120 min. Plasma membrane proteins were isolated by cell surface biotinylation and LMTK2 abundance in each domain was assessed by WB. LMTK2 expression in whole cell lysates (WCL) was used as loading control. 3–7 experiments/group. Additionally, TGF-β1 treatment for 60 min increased the apical membrane LMTK2 in HBE (C,D) and F508del HBE cells (F,G) without affecting the basolateral membrane LMTK2. LMTK2 expression in WCL was used as a loading control. Five replicates from three HBE cell donors (C,D) and three replicates from one F508del HBE cell donor (E,F). qRT-PCR experiments demonstrating that TGF-β1 did not increase the LMTK2 mRNA level in HBE cells (G). TGF-β1 (15 ng/ml) was added to the basolateral medium and cells were incubated for 6, 12 or 24 h. Raw data were analyzed using the ΔΔCt method. Changes in the LMTK2 mRNA were normalized to GAPDH and expressed as Log2 FC versus untreated cells (time zero). Experiments were performed in triplicates in HBE cells from 4 lung donors. ^∗p < 0.05 and ^∗∗p < 0.01 versus vehicle. Error bars, SEM. AP, apical; BL, basolateral; BT, biotinylation; IB, immunoblot; WCL, whole cell lysate.