Mammalian expressional plasmid pcdna3.1 his v5 topo

Human lung fibroblast cell lines (Mrc5 and IMR90) were purchased from ATCC (Manassas, VA). Human primary lung fibroblasts (HLF) and lung myofibroblasts (IPF-LF) from adult normal human subjects and IPF patients were obtained from the Center for Organ Recovery and Education and Lung Transplantation at the University of Pittsburgh. The study was approved by the institutional Review Board at the University of Pittsburgh (STUDY18100070). Cells were cultured in Eagle’s Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS) in 5% CO2 cell culture incubator. V5 antibody, mammalian expressional plasmid pcDNA3.1/His-V5-topo, and Escherichia coli Top10 competent cells were purchased from Life technologies (Grand Island, NY). Nedd4L, TβRII, collagen I, ubiquitin, smurf1, Smad4, p-Smad2/3, and Smad2/3 antibodies were purchased from Cell Signaling (Danvers, MA). Fibronectin (FN), alpha-smooth muscle actin (α-SMA), E2F4 antibodies, immobilized protein A/G beads, and control IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HLM006474, bleomycin, and β-actin antibody were purchased from Sigma (St. Louis, MO). Human recombinant TGF-β1 was purchased from R&D systems (Minneapolis, MN). All materials used in the experiments are the highest grade commercially available.

Murine lung epithelial (MLE12) cells (from ATCC) were cultured with HITES
medium containing 10 % fetal bovine serum (FBS) at 37°C in a 5 %
CO₂ incubator. Mammalian expressional plasmid
pcDNA3.1/His-V5-topo, Escherichia coli Top10 competent cells,
and V5 tag antibody were from Life Technologies (Grand Island, NY). FBXL19
antibody was purchased from Abgent (San Diego, CA). Cycloheximide, trichostatin
(TSA), β-actin antibody, FBXW17 shRNA, and control shRNA were from Sigma
(St. Louis, MO). MG-132 was from EMD Chemicals (Philadelphia, PA). Immunobilized
protein A/G beads and control IgG were from Santa Cruz Biotechnology (Santa
Cruz, CA). Myc tag and Rac1 antibodies were from Cell Signaling Technologies
(Danvers, MA). All materials in the highest grades used in the experiments are
commercially available.

Human lung microvascular endothelial cells (HLMVECs, from ATCC) were cultured with endothelial growth medium-2 (EGM-2) containing antibiotics at 37°C in 5% CO₂. Human hybridoma endothelial cells (EAhy 926, ATCC) and HEK293 cells (from ATCC) were cultured in DMEM medium containing 10% fetal bovine serum (FBS). V5 tag antibody, mammalian expressional plasmid pcDNA3.1/His-V5-topo, and Escherichia coli Top10 competent cells were purchased from Life technologies (Grand Island, NY). ISG15, p65, ubiquitin, and WIP1 antibodies were purchased from Cell Signaling (Danvers, MA). ICAM1 and VCAM1 antibodies, immobilized protein A/G beads, and control IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FBXL19 antibody was from Abgent (San Diego, CA). Lipopolysaccharide (LPS), T7 tag, and β-actin antibodies were purchased from Sigma (St. Louis, MO). Human recombinant TNFα was purchased from R&D systems (Minneapolis, MN). All materials used in the experiments are the highest grade commercially available.