Monoclonal antibody against β actin

Cell lysates and supernatants from DCs culture were subjected to SDS-PAGE analysis and western blotting. The proteins were resolved on a 15% SDS-PAGE gel, and transferred to nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden). Membranes were blocked for 1 h in TBS (0.1% Tween-20; 5% non-fat dry milk) and incubated with primary antibodies at 4°C, overnight. Primary antibody used was mouse monoclonal against the p20 subunit of caspase-1 (Adipogen, San Diego, CA, United States). Monoclonal antibody against β-actin (Cell Signaling Technology, Danvers, MA, United States) was used as a loading control blot (1:1,000). The membranes were washed three times for 10 min in TBS with 0.1% Tween 20. Next, membranes were incubated for 1 h at room temperature with the suitable HRP-conjugated secondary antibody (1:1,000). Immunoreactive bands were visualized using Luminol chemiluminescent HRP substrate (Millipore).

Lung tissues and cells were homogenized in lysis buffer with protease inhibitor cocktail (Roche, IN, USA)^{25 (link)}. After determining the protein concentration by DC protein assay kits (Bio-Rad, Hercules, CA, USA), total soluble proteins (20 μg) were separated on 4–12% gradient gels (Invitrogen, CA, USA)^{25 (link)}. The proteins were transferred to a nitrocellulose membrane (Amersham, Little Chalfont, UK). Nonspecific binding was blocked with 5% skim milk (BD, CA, USA) at room temperature for 1 h^{25 (link)}. Then, the protein blots were probed with specific primary antibodies (1:1000 dilutions) against phospho-Smad1/5 (Cell Signaling, Frankfurt, Germany), phospho-Smad2 (Millipore, MA, USA), phospho-Smad3 (Millipore, MA, USA), PECAM-1 (Millipore, MA, USA), and L- and S-endoglin (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. After incubating with host-specific secondary antibodies (goat anti-rabbit IgG-HRP) for 1 h at room temperature, the immunoreaction was detected with a chemiluminescent substrate kit (Thermo Scientific, Rockford, IL, USA) and quantitated by densitometric scanning of the X-ray film with SLB MyImager (UVP Inc, Upland, CA, USA)^{25 (link)}. The blots were normalized for protein loading by washing in stripping solution and reprobing with a monoclonal antibody against β-actin (Cell Signaling, Frankfurt, Germany)^{25 (link)}.

Whole cell extracts from cultured cells were prepared using RIPA Lysis and Extraction Buffer containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Rockford, IL). Protein quantification was done using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Samples were prepared with NuPAGE LDS sample buffer and NuPAGE® Sample Reducing Agent. Total protein (20–35 μg) was loaded onto NuPAGE® Novex 4–12% Bis-Tris Plus Gels with MES SDS Running Buffer with NuPAGE® Antioxidant and separated by XCell SureLock™ Mini-Cell Electrophoresis System (Thermo Fisher Scientific). Protein was transferred to iBlot® 2 Dry Blotting System and immunoblotting were carried out according to standard protocols. Monoclonal antibody against COMT was purchased from Abcam (#ab185954), whereas antibody for CASP3, cleaved CASP3, cleaved BID, XIAP, and cIAP2 were purchased from Cell Signaling Technology (Danvers, MA). Monoclonal antibody against β-Actin (Cell Signaling Technology) and GAPDH (Santa Cruz) were used to confirm equal loading. Protein complexes were visualized with Image Studio™ Software (LI-COR, Lincoln, NE) using the Odyssey® CLx Imaging System (LI-COR). The raw blotting images are available in S1 Fig.