Objective Fibrotic changes are initiated early in acute respiratory distress syndrome. In separate experiments, A549 cells were incubated with medium, AdHSP, or AdGFP. Some cells were also stimulated with tumor necrosis factor-. After 48 hrs, cytosolic and nuclear proteins from rat UTP14C lungs or cell cultures were isolated. These were subjected to immunoblotting, immunoprecipitation, electrophoretic mobility shift assay, fluorescent immunohistochemistry, and Northern blot analysis. Measurements and Main Results Alveolar type I cells were lost within 48 hrs of inducing acute respiratory distress syndrome. This was accompanied by alveolar type II cell proliferation. Treatment with AdHSP preserved alveolar type I cells and limited alveolar type II cell proliferation. Heat shock protein 70 prevented overexuberant cell division, in part, by inhibiting hyperphosphorylation of the regulatory retinoblastoma protein. This prevented retinoblastoma protein ubiquitination and degradation and, thus, stabilized the interaction of retinoblastoma protein with E2F1, a key cell division transcription factor. Conclusions Heat shock protein 70-induced attenuation of cell proliferation may be a useful strategy for limiting lung injury when treating acute respiratory distress syndrome if consistent in later time points. for 30 mins at 4C. The supernatants (cytoplasmic fraction) were collected for immunoprecipitation and immunoblotting as described below. Immunoblot Analysis A total of 30 g of total protein lysate was separated on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblotting as previously described (21). All signals were detected by enhanced chemiluminescence and quantified by scanning densitometry. DNA polymerase- and DNA polymerase- were identified with primary polyclonal rabbit DNA polymerase- and polymerase- antibodies (Santa-Cruz Biotechnology, Santa-Cruz, CA). E2F1 was identified using a primary mouse monoclonal antibody (Lab Vision, Fremont, CA). Rb was identified with a primary mouse monoclonal Rb antibody (Cell Signaling Technology, Beverly, MA). Phospho-Rb (pRb) was identified with a primary polyclonal rabbit pRb antibody (Cell Signaling Technology). Hsp70 was identified using a mouse monoclonal antibody (StressGen Biotechnologies, Victoria, BC, Canada). In all cases, the secondary antibody was mouse anti-rabbit immunoglobulin G or goat anti-rabbit immunoglobulin G (Jackson, Immunoresearch, West Grove, PA). Electrophoretic Mobility Shift Assay of E2F1 DNA Binding Activity Electrophoretic mobility shift assay was performed as Poziotinib supplier previously described (27, 30). Briefly, a double-stranded DNA oligonucleotide containing a consensus -E2F1 binding site was labeled with 32P. The labeled oligonucleotide was purified on a G-25 Sephadex column. Nuclear extracts containing 10 g of nuclear protein were incubated with binding buffer (20 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [Hepes; pH 7.9], 40 mM KCl, 6 mM MgCl2, 1 mM phenylmethyl sulfonyl fluoride, 1 mM dithiothreitol, 0.5% nuclear protein-40), deoxyinosinic deoxycytidylic acid (1 g/L) for 20 mins at room temperature. The Poziotinib supplier labeled oligonucleotide was added for 20 mins. Specificity of DNA binding was determined by cold competition. Immunoprecipitation To demonstrate that prevention of Rb phosphorylation resulted, in part, from an interaction with Hsp70, we immunoprecipitated nuclear protein extracts with an antibody to Rb and performed immunoblotting with antibodies to Hsp70 and E2F1. Samples containing 500 g of cytosolic extract were immunoprecipitated using mouse monoclonal Rb antibody (Cell Signaling Technology), diluted 1:100. Samples were agitated overnight at 4C. Protein A/G beads (Sigma) were added, and the samples were agitated for 1 hr at 4C and centrifuged Poziotinib supplier at 14,000 rpm for 5 mins at 4C. The pellet was washed three times with lysis buffer (31), suspended in sample buffer, and boiled at 95C for 5 mins. The resulting mixture was prepared for immunoblotting. As controls for the specificity of immunoprecipitation, we incubated the lysate from 2CLP AdHSP and 2CLP PBS nuclear lung extracts overnight at 4C with an amount of mouse immunoglobulin G the same.