Bicinchoninic acid bca protein assay

Dissected lung tissues were placed in cell lysis buffer freshly supplemented with protease inhibitor cocktail (Sigma, Alexandria, VA, USA) and phosphatase inhibitors (Roche, Nutley, NJ, USA) and homogenized for protein extraction. The protein samples were quantitated with BCA (bicinchoninic acid) protein assay (Beyotime). Protein samples (20–50 μg) were resolved on a 10% SDS–polyacrylamide gel and transferred onto PVDF membranes (Roche, 3010040001). The membranes were blocked with 5% skimmed milk in Tris-buffered saline (TBS) at RT on a shaker for 1h and then incubated with the specific primary antibodies: Collagen I (Meridian, Beijing, China; no. 1:1000), α-SMA (Abcam, no. ab9588, 1:1000), and α-tubulin (Beyotime, AF0001 1:10,000) overnight at 4 °C. After washing with TBS-T buffer, the membrane was incubated with proper HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibody (1:10,000) at RT for 1h before detection by ECL reagent (Enhanced Chemiluminescence, Amersham, UK) and image acquisition.

Total protein lysates were prepared from HCC cells and tissues in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, and 5 mM protease inhibitor cocktail; pH 7.4), and the protein concentration was measured by a BCA (bicinchoninic acid) protein assay (Beyotime, Inc., Shanghai, China). Equal amounts of protein lysates were separated on 10% SDS-PAGE at 100 mV for 2 h. Then, the separated proteins were transferred onto PVDF membranes at 80 mV for 1 h. The blots were first blocked with 5% nonfat milk, followed by incubation with primary antibodies overnight at 4 °C. The blots were then incubated with HRP-conjugated secondary antibody at room temperature for 1 h. Then, the blots were developed by an ECL chemiluminescence method and the specific protein bands were quantified with ImageJ software. β-Actin was used as an internal control. Antibodies against HSF1, caspase 3, caspase 9, BAD, Bcl-2, Bcl-xL, BIM and BID were purchased from Santa Cruz (Dallas, Texas, USA). Antibodies against β-actin, HSP90, HSP60, SMAC and Apaf-1 were purchased from Origene (Rockville, MD, USA).

Spinal cord tissue from T7 to T10 was collected at a specific time after surgery. Briefly, the spinal cord tissue and HAPI cells were lysed using RIPA with phosphatase inhibitor and protease inhibitor cocktail and then the concentration of protein was measured using the BCA (Bicinchoninic Acid) protein assay (Beyotime, Shanghai, China); equivalent amounts of protein were separated using 8%‐12% SDS–PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Massachusetts, USA). After blocking with 5% non‐fat milk for 2 hours, the primary antibodies were incubated: anti‐BRD4 (1:1000), p38 (1:1000), p‐p38 (1:1000), JNK (1:1000), p‐JNK (1:1000), ERK (1:1000), p‐ERK (1:1000), anti‐p65 (1:1000), anti‐p‐p65 (1:1000), anti‐IκBα (1:1000), anti‐INOS (1:1000), anti‐COX‐2 (1:1000), anti‐β‐actin（1:1000） followed by incubation with the respective secondary antibodies for 60 minutes. The resultant signals were detected using the ChemiDicTM XRS +Imaging System (Bio‐Rad) and the intensity of these bands was analysed using Image Lab 3.0 software (Bio‐Rad).