α tubulin

At the end of treatment with PM_2.5 in the absence and presence of PAI-1 inhibitor TM5614, supernatants from control and treated wells were collected. The cell lysates were prepared using RIPA lysis buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisher Scientific) containing protease inhibitor cocktail (cOmplete) and phosphatase inhibitor cocktail (PhosSTOP) (Sigma). Cell lysates were pooled from 3 wells for each control and treatment group and equal amounts of protein were subjected to gel electrophoresis, transferred to PVDF membranes, and subjected to western blotting using antibodies against PAI-1 (Molecular Innovations, Inc.), type I collagen (Southern Biotech), fibronectin (Millipore), sterol regulatory element binding protein-1 and 2 (SREBP1, SREBP2), nuclear factor erythroid related factor 2 (Nrf2) (Abcam), and α-tubulin (GenScript), with HRP-tagged corresponding secondary antibodies. The membranes were developed with enhanced chemiluminescence reagents (Luminata Forte, Millipore, Billerica, MA), and images of protein bands were captured using a BIO-RAD ChemiDoc XRS system (BIO-RAD, CA).

Frozen heart or kidney tissues were submerged in tissue lysis buffer supplemented with protease inhibitor cocktail (cOmplete) and phosphatase inhibitor cocktail (PhosSTOP) (Sigma). Tissue lysates were prepared by homogenization using Pestle Mixer (Argos Tech.) and centrifugation at 10,000 rpm for 15 min at 4 °C. The supernatant was used as source of protein. Equal amount of heart or kidney tissue lysates from each mouse were pooled (n = 5) and loaded on 4–12% gradient gel. Equal amount of heart lysate proteins from each mouse (n = 4–5/group) were also subjected to western blot analysis. The proteins were transferred to PVDF membranes and processed for immunoblot analysis using antibodies against integrin αV, integrin β3 (Abcam), CBX7 (Abcam), Nrf2 (Abcam), pERK1/2, p-p38 (Cell Signaling) and α-tubulin (GenScript). For coupled immunoprecipitation and immunoblot assay, 1 mg of total heart tissue lysates pooled from 5 mice (200 μg/mouse) were immunoprecipitated with PAI-1 antibody (Molecular Innovations) and Protein A-Agarose beads overnight at 4 °C. The beads-bound proteins were spinned down and washed with lysis buffer for three times and beads were resuspended in LDS loading buffer (Invitrogen), denatured at 95 °C and loaded on 4–12% gradient gel and subjected to western blot using antibodies against integrin αV, integrin β3, protein phosphatase 1 (PP1), and PAI-1.