Western blotting chemiluminescence luminol reagent

Lung and liver tissues were excised and homogenized, and the total cellular proteins were extracted by ice-cold RIPA lysis buffer (150 mmol/liter of NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris (pH 7.4)) containing protease inhibitors. The lysates were centrifuged at 12,000×g for 20 min at 4 °C. Soluble proteins (20 µg) were separated by 8 ~ 12% SDS-PAGE gel electrophoresis and immunoblotting was conducted with primary antibodies against AHR (#67,785; Proteintech Group, AL, USA; 1:3000), CAR (#ab186869; Abcam, Cambridge, UK; 1:1000), PXR (#67,912; Proteintech Group, AL, USA; 1:3000), and β-actin (#60,008; Proteintech Group, AL, USA; 1:4000), followed by HRP-conjugated secondary antibodies (Beyotime Biotech Inc., Nantong, China; 1:3000). Immunolabeling was visualized via incubating the bands with Western Blotting Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, CA, USA). The density of the specific protein bands was quantified using ImageJ software. n = 3.

Cell lysates were prepared using 2X SDS gel electrophoresis sample buffer. Proteins were separated by 4–20% gradient Tris-glycine gel (Invitrogen, Carlsbad, CA) electrophoresis, and Immobilon-P PVDF membrane (Millipore) transfer was performed as previously described [2 (link), 9 (link)]. Membranes were blocked in 5% dry milk-PBST (0.1% Tween 20 in PBS) and probed with primary antibodies at dilution of 1:1000. The primary antibodies used were as follows: pRB antibody (Cell Signaling), P-pRB (Cell Signaling), p53 (B-P3, Santa Cruz), p-p53 S15 (Cell Signaling), p16 (N-20, Santa Cruz), Myosin Light Chain 2 (MLC2; Cell Signaling), P-MLC2 (Thr18/Ser19; Cell Signaling), or P-cofilin (Cell Signaling). The membranes were then probed with anti-rabbit IgG or anti-mouse IgG secondary antibodies conjugated to HRP (Santa Cruz Biotechnology). The membranes were visualized by using Western Blotting Chemiluminescence Luminol Reagent (Santa Cruz). As a loading control, membranes were probed with an antibody specific for β-actin (Sigma-Aldrich, St. Louis, MO) at 1:10,000. Membranes were stripped using Restore Western Blot Stripping Buffer (Pierce, Rockford, IL).

Antibodies against phospho-mTOR, total and phospho-Akt (p-Akt), phospho-cAMP response element binding protein (p-CREB), phospho-ERK1/2 (p-ERK1/2), total and phospho-p38 (p-p38), phospho-Src (p-Src), cleaved caspase-3, caspase-3, and poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Danvers, MA, USA). Western blotting chemiluminescence luminol reagent was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Direct-zol™ RNA MiniPrep Plus mRNA extraction kit was purchased from Zymo Research (Irvine, CA, USA). SsoAdvanced™ Universal SYBR^® Green Supermix and iScript^TM Reverse Transcription Supermix were purchased from BioRad Laboratories (Hercules, CA, USA). Primers for VCAM-1 (forward ACA-GAA-GAA-GTG-GCC-CTC-CAT and reverse TGG-CAT-CCG-TCA-GGA-AGT-G) and GAPDH (forward GTC-TCC-TCT-GAC-TTC-AAC-AGC-G and reverse ACC-ACC-CTG-TTG-CTG-TAG-CCA-A) were purchased from Integrated DNA Technologies (Coralville, IA, USA). Primers for ERK2 and centromere protein F (Cenpf) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Milliplex Map Akt/mTOR Phosphoprotein Magnetic Bead 11-Plex Kit-Cell Signaling was purchased from Millipore (Billerica, MA, USA).