Ab10739

Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 150 mM NaCl), supplemented with protease inhibitor cocktail (Roche), 5 mM NaF and 0.2 mM Sodium orthovanadate. Protein lysates were mixed with reducing agent and LDS sample buffer (Novex, ThermoFisher) and denatured at 70°C for 10 min and loaded in Novex Nupage 4–12% Bis-Tris gels. Gels were transferred onto nitrocellulose membrane using the iBlot2 system (ThermoFisher). Membranes were blocked with PBS-0.05% Tween and 5% milk for 1 h at room temperature with agitation, before overnight incubation with primary antibodies. Antibodies against PKR (ab45427 Abcam), ILF3 (ab92355 Abcam), s6RP (2317S CST), eIF6 (3833S CST), ILF2 (ab154169 Abcam), α-tubulin (CP06 Merck), fibrillarin (ab5821 Abcam), phospho-eIF2α (Ser-51) (D9G8) (3398S CST), IFNAR1 (ab10739 Abcam), IFIT3 (ab76818 Abcam), OASL (ab191701), IRF1 (CST #8478), eIF3M (Bethyl, A305–029A), anti-rabbit HRP (CST) and anti-mouse HRP (Bio-Rad) were used. Proteins were visualized using ECL (Pierce) on a Bio-Rad ChemiDoc imaging system. Protein bands were quantified using ImageJ (v1.51p) software and normalized to α-tubulin or fibrillarin.

For type I and III interferon (IFN) stimulations, basolateral region of hBECs were treated with recombinant human (rh) IFN-β protein (8499-IF; R&D Systems) using 0.1, 1, 10, 100, 1000 U/mL and recombinant human (rh) IL-29/IFN-λ1 protein (1598-IL; R&D Systems) using 0.1, 1, 10, 100 ng/mL for 24 h, respectively. To validate IFN stimulation, the induction of interferon-stimulated genes (ISGs) were analyzed by qPCR. To block type I IFN signaling experiment, 2 μg/mL of IFNAR1 (ab10739; abcam, 1:50) and normal goat IgG control antibody (AB-108-C; R&D Systems, 1:500) were treated with basolateral region for 1 h prior to the rhIFN-β protein treatment. Similarly, to block type III IFN signaling experiment, 10 μg/mL of IFNλR1 (PBL assay science; 21885-1, 1:50), mouse IgG isotype control antibody (31903; Invitrogen, 1:500) were treated with basolateral region for 1 h prior to the rhIFN-λ1 protein treatment.