Sc 2060

Cells were collected and lysed using NP40 lysis buffer for 30 minutes on ice with constant agitation. Lysates were centrifuged at 12,000 x g for 5 minutes at 4°C, supernatants were collected, and then protein content was quantified (#500-0006, Biorad, Hercules, CA, USA). Protein was separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk, then probed with primary antibodies against Lamin A (MAB3540, Millipore, Billerica, MA, USA), Progerin (#05-1231, Millipore), GFP (AB3080, Millipore), Oct4 (MAB4401, Millipore), and Notch2 (Ab8926, Abcam, Cambridge, MA, USA) in 5% milk for 1h at room temperature. Membranes were then washed in TBS-tween and incubated in 5% milk supplemented with HRP-linked secondary antibodies (SC2005, goat anti-mouse IgG-HRP; SC2060, goat anti-mouse IgG1-HRP; SC2004, goat anti-rabbit IgG-HRP; SC2020, donkey anti-goat IgG-HRP, Santa Cruz Biotechnology, Dallas, Texas, USA). Protein bands were detected by enhanced chemiluminescence using the ECL Plus kit (RPN2132, GE Life Sciences, Pittsburg, PA, USA). Total protein stained with coomassie brilliant blue (Biorad, 161-0400) was used as a loading control.

NHBE cells grown at ALI were lysed with RIPA buffer and protease inhibitors. Cell lysates underwent immunoprecipitation (IP) for 2 h with primary anti eNOS MAB (BD Transduction, #610296, 1:500) or with primary anti Hbβ monoclonal antibodies (MAB; AbCam, #Ab100952, 1: 500), washed 3 × in PBS, extracted from beads with Laemmli buffer, put in 2 × loading buffer, and run on a 4–15% SDS-page gel,. The membrane was incubated overnight with primary antibodies. We used primary anti Hbβ MAB (AbCam, #Ab100952, 1:1000) with secondary (Santa Cruz, SC-2061, 1:3000); primary anti eNOS MAB (BD Transduction, #610296, 1:1000) with secondary (Santa Cruz, SC-2060, 1: 3000). Blots were visualized using the Biorad Chemidoc system.

Cell pellets corresponding to OD₆₀₀ 5–10 were protein extracted using the alkali extraction protocol described in [18 (link)], and samples corresponding to OD₆₀₀ 0.5–1 were analyzed by SDS-PAGE and western blotting (WB). Monoclonal mouse anti-His primary antibody (Sigma H1029-2ML, 1:5000 dilution) and anti-mouse-HRP secondary antibody (Santa Cruz Biotechnology sc-2060, 1:10,000 to 1:20,000 dilution), or polyclonal rabbit anti-NifU ([13 (link)], 1:5000 dilution), polyclonal rabbit anti-NifS ([13 (link)], 1:200 dilution), polyclonal rabbit anti-NifM (1:4000 dilution), and anti-rabbit-HRP secondary antibody (Sigma A0545-1ML, 1:10,000 dilution) were used.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in [19 (link)].
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.