Western Blot data for Shhcre;Lonp1 at E13.5
Description
Raw gel images for western blot results. The corresponding protein targets were shown in the title of each image. Corresponding to Figure 1A of manuscript "Context-Dependent Roles of Mitochondria in Orchestrating the Balance between Airway Progenitor versus Progeny Cells". Embryonic lungs at E13.5 were collected for protein extraction. Equal amount of proteins were loaded onto gel. Three biological replicates of the same litter were analyzed for each genotype in the order of control and Shhcre;Lonp1 mutant.
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Embryonic lungs at E13.5 were collected in RIPA Lysis and Extraction Buffer (9806, CST) supplemented with cOmplete Protease Inhibitor Cocktail tablets (11697498001, Roche). Samples were homogenized in a QIAGEN TissueLyser II and proteins were purified from the supernatant after high-speed centrifugation. Protein concentrations were measured by Pierce BCA Protein Assay kit (23225, Thermo Scientific). 10 µg protein sample was loaded in each well and run on a 4%–12% SDS-PAGE gel (NP0321BOX, Invitrogen), then transferred to the nitrocellulose membrane (1620112, 0.2 µm, Bio-Rad) by the Trans-Blot Turbo Transfer System (Bio-Rad). Then the membranes were blocked in TBST (0.1% Tween-20) with 5% BSA for 1.5 hours at room temperature (RT) before incubation in primary antibodies overnight at 4°C. After 3 rounds of washing with TBST, membranes were incubated in the secondary antibody for 1 hour at RT. Images were acquired by Odyssey DLx Imager (LI-COR) and quantified by ImageJ. The following primary antibodies were used: LONP1 (28020, CST, 1:1000), NDUFA9 (459100, Invitrogen, 1:1000), SDHA (5839S, CST, 1:1000), UQCRB (10756-1-AP, Proteintech, 1:1000), COX I (ab14705, Abcam, 1:500), COX II (55070-1-AP, Proteintech, 1:1000), ATP5A1 (14676-1-AP, Proteintech, 1:500) and beta-Actin (Novus Biologicals, NB600501, 1:5000). Secondary antibodies used were: donkey anti-Mouse IgG IRDye 680RD (LI-COR, 926-68072, 1:10000) and donkey anti-Rabbit IgG IRDye 800CW (LI-COR, 926-32213, 1:10000). Three biological replicates were analyzed for each condition or genotype.