Goat anti hes1

The protein content of cell extracts or tissue prepared in radioimmunoprecipitation assay lysis buffer was determined using the Bradford protein assay kit (Bio-Rad). About 30 μg of proteins were separated by SDS-polyacrylamide gel electrophoresis. After transferring to nitrocellulose membranes, immunoblots were probed separately with various primary antibodies. Subsequently, the immunoblots were blocked with 5% skimmed milk in Tris-buffered saline solution. Fluorescently labeled or horseradish peroxidase–conjugated secondary antibodies were detected by the Odyssey Infrared Imaging System (LI-COR Biosciences). Primary antibodies were used as following: rabbit anti-JMJD3 (Millipore; catalog no.: 07-1533), rabbit anti-H3K27me3/me2/me1 (Abclonal; catalog nos.: A2363, A2362, and A2361), rabbit anti-UTX (GeneTex; catalog no.: GTX12146), rabbit anti-eNOS (Cell Signaling Technology; catalog no.: 32027), rabbit anti-VE-cadherin (Santa Cruz; catalog no.: sc-28644), mouse anti-α-SMA (Sigma; catalog no.: A5228), goat anti-Hes1 (Santa Cruz; catalog no.: sc-13844), rabbit anti-PCNA (Santa Cruz; catalog no.: sc-7907), mouse anti-β-actin (GeneTex; catalog no.: GTX629630), and mouse anti-GAPDH (Santa Cruz; catalog no.: sc-32233).

Differentiation was quantified in ALI cultures using previously reported methods and antibodies (70 (link)). Ciliated and goblet cell differentiation intermediates were defined as previously reported (36 (link)) and are illustrated in (Supplemental Figure 2).Three individuals, who were blind to the groups, used guide images to classify ciliated and goblet cell differentiation intermediates, and the 3 values were averaged. For analysis of cell surface markers, cultures were fixed with 10% neutral buffered formalin and blocked with 5% bovine serum albumin/1× PBS. Additional antibodies used in this study were as follows: mouse anti-JAG1 C-terminal–specific antibody (BD Biosciences, 612346, 1:50); rabbit anti-JAG2 N-terminal–specific antibody (Cell Signaling Technology [CST], C23D2, 1:50); rabbit anti-JAG2 C-terminal–specific antibody (CST, C83A8, 1:50); goat anti-HES1 (Santa Cruz Biotechnology, sc-13842, 1:40); rabbit anti-RBPJ (MilliporeSigma, AB5790, 1:100). Primary antibodies were detected with Alexa Fluor 488– or Alexa Fluor 594–labeled secondary antibodies (Jackson ImmunoResearch, 1:500). Nuclei were detected with DAPI.