Af1210

Vesicle samples were lysed in 10× cell lysis buffer (Cell Signaling Technology, catalog no. 9803). Western blot was performed as described previously (62 (link)). To detect protein expression of Ghr, exosome cargo protein TSG101 (involved in multivesicular biogenesis), CD63, and CD81 (tetraspanins), 50 μg of exosome lysate was run through NuPAGE 4 to 12% bis-tris gel (Thermo Fisher Scientific, Waltham, MA) and blotted onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Waltham, MA). Polyclonal antibody to Ghr (Boster Biological Technology, PA1726; R&D, AF1210), monoclonal antibody to Ghr (sc-137185, Santa Cruz Biotechnology; ab134078, Abcam), TSG101 (Ab125011, Abcam), CD63 (Ab68418, Abcam), and CD81 (catalog no. 10630D, Thermo Fisher Scientific, Waltham, MA), as well as associated horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology), were used as per the manufacturer’s instructions. The bands were imaged and analyzed using the ECL system (Bio-Rad, Hercules, CA).

At the time of tissue collection, mice were euthanized by intraperitoneal injection of a mixture of ketamine (100 mg/kg; Fort Dodge) and xylazine (10 mg/kg; Shenandoah). The chest cavity was exposed, and the lungs were cleared of blood by perfusion with cold phosphate-buffered saline (PBS) via the right ventricle. Lungs were inflated with 10% formaldehyde under constant pressure and allowed to fix for 16 hours. Tissues were then prepared for optimal cutting temperature (O.C.T.) compound embedding or paraffin embedding. Cryosections were used for immunofluorescent detection and staining. Antibodies used in immunofluorescent staining were Sftpc (Goat and Rabbit; 1:200; B. Stripp laboratory), COL1A1 (NB600-408; 1:200; Novus Biologicals), HTII-280 (1:200; a gift of the L. G. Dobbs laboratory, University of California, San Francisco, San Francisco, CA), CD31 (1:200; mouse, 303101, BioLegend), GHR (Goat, AF1210; 1:200; R&D Systems), red fluorescent protein (Rabbit; 1:200; 600-401-379, Rockland), and correspondent secondary antibodies conjugated to Alexa 546 or 488 (1:500; A-21085, A-11056, and A-11008, Thermo Fisher Scientific). Paraffin sections were used for hematoxylin and eosin (H&E) and Masson’s trichrome staining.