Carbobenzoxy leu leu leucinal

Primary hepatocytes were treated with TGF-β1 for 6 h and then lysed in a buffer, as described in a previous study^{19 (link)}. For the ubiquitination assay, the hepatocytes were first transfected with shRNA adenovirus or plasmids using effectene transfection reagent (Qiagen, Germany) for 24 h, stimulated with TGF-β1 for 2 h, and finally treated with carbobenzoxy-Leu-Leu-leucinal (MG132, Sigma, USA) for 4 h. The lysates were incubated with antibodies against Smad3, p-Smad3 or p-Smad2 overnight, and then protein A/G beads (Roche, Switzerland) were added for 2 h. The precipitates were boiled for 5 min and were examined using immunoblot with mouse anti-Ets-1 (Santa Cruz, USA) or mouse anti-ubiquitin antibody (CST, MA, USA).

An ROS scavenger, N-acetyl-l-cysteine (NAC; catalog no. A7250), and a proteasome inhibitor, carbobenzoxy-Leu-Leu-leucinal (MG132, catalog no. M8699) were purchased from Sigma-Aldrich. A549 and MRC-5 cells were treated with different concentrations of MG132 or NAC, and the cytotoxicity was determined by WST-1 assay at 48 h posttreatment. NAC at 15 mM and MG132 at 0.05 μM were found to be nontoxic to the cells. To understand the effects of NAC and MG132, cells were infected with IAV strain PR8 at an MOI of 0.01 and NAC or MG132 was added in the overlay media. Supernatants were collected at 45 hpi, and virus titers were determined by plaque assay.