Polyvinylidene fluoride (pvdf)

Protein extracts (50 μg) were run on 10% SDS‐PAGE. The protein was then transferred to a polyvinylidene difluoride membrane (PVDF, Amersham Biosciences). The membrane was blocked for 1‐hour at room temperature with 10% BSA in phosphate‐buffered saline/0.05% Tween 20. The blots were incubated overnight at 4°C with anti‐α‐SMA, anti‐collagen IV, anti‐fibronectin, anti‐phosp‐IκB, anti‐IκB, anti‐NRF, anti‐p65, anti‐p50, anti‐Histone H1 or anti‐Tubulin antibody and secondary antibody (Cell Signalling, Danvers, MA). The protein expression was visualized using enhanced chemiluminescence reagents (Bio‐Rad, Hercules, CA). The amounts of the proteins were analysed using Image J analysis software.

Cells were lysed in lysis buffer and centrifuged at 12,000 rpm for 10 min. The protein samples in the supernatant were immediately collected, and the concentration was measured using the Bradford method (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred onto PVDF (polyvinylidene fluoride) membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). After blocking with 5% nonfat dry milk in PBS containing 0.1% Tween-20 for 2 h at room temperature, the membranes were incubated at 4 °C overnight with primary antibodies (1:1000) against cyclinD1 (#2978, cell signaling), CEBP/β(#3087, cell signaling), and β-actin (sc-47778, Santa Cruz) and subsequently incubated with secondary horseradish peroxidase antibody. The immunoreactive proteins were visualized using an enhanced chemiluminescent reagent (Millipore Corporation, USA).

THP-1-derived macrophages were treated or not with different concentrations of PpSP32 (0.5 μg/ml, 2 μg/ml, and 5 μg/ml) for 48 h at 37 °C, 5% CO_2, then stimulated with 100 ng/ml of LPS for 3 h at 37 °C, 5% CO_2. Total cell lysates were extracted at room temperature with 100 µl of Laemmli buffer (1x) per 5 × 10⁵ cells. Protein concentration was determined by using the Bicinchoninic Acid Protein Assay Kit, (BCA, Sigma) with bovine serum albumin (BSA) as standard. Whole-cell lysates (30 µg/lane) were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinyl difluoride membrane (PVDF, Amersham). After washing, the membrane was incubated with anti-phospho I kappa B alpha (anti-pIκB-α) antibody (Cell Signaling Technology) at 1:2000 overnight or anti-β actin at 1:1000 (Cell Signaling Technology) for 2 h at room temperature. After washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG HRP at 1:2000), immunoblots were determined by enhanced chemiluminescence.

Proteins were extracted from hamster liver tissues (120 mg) using RIPA buffer (50 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) and protein concentration was determined using the Bradford assay. Ten micrograms of liver protein were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF, Amersham, Piscataway, NJ, USA) for 2 h at 60 V. After blocking with 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 (PBST), membranes were incubated with appropriate primary antibodies overnight at 4°C and then incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. Chemiluminescent reaction was developed using ECL western blotting detection reagent (GE Healthcare) and then captured using the ImageQuant LAS4000 mini imager (GE Healthcare). The ImageQuant TL software v2005 (1.1.0.1) (Non-linear Dynamics, Durham, NC, USA) was used for densitometry of each band. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

The hippocampus was homogenized in 500 μl RIPA buffer (Thermo Fisher Scientific, Waltham, MA, United States) with Pro-Prep^TM (iNtRON Biotechnology) and equalized to the same amount (20 μg) of protein. The equalized samples were separated with 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories, Inc., Hercules, CA, United States), and separated proteins were transferred to a PVDF (Amersham Biosciences, Piscataway, NJ, United States). Membranes were blocked in 5% skim milk (Bio-Rad Laboratories, Inc.) solution for 1 h at room temperature and incubated with primary antibody overnight at 4°C: actin (Dilution ratio 1:2,000), BDNF (Dilution ratio 1:1,000), CREB (Dilution ratio 1:1,000), and pCREB antibodies (Dilution ratio 1:1,000). Membranes were subsequently incubated with appropriate secondary mouse and rabbit antibodies (Cell Signaling Technology) for 1 h at room temperature. Actin was used as a loading control for all experiments. The density of the protein band was quantified using an ImageQuant LAS 4000 mini (Fujifilm, Tokyo, Japan).

Protein samples for the cap-affinity assay were separated by 8-15% gradient SDS-PAGE (polyacrylamide gel electrophoresis). Ten or twelve percent SDS-PAGE was used for PARP and eIF4E proteins, respectively. Following protein transfer to PVDF (Amersham Biosciences) the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). The membranes were then incubated for 1 hour at ambient temperature or overnight at 4°C with the chosen primary antibody. The primary antibodies employed were rabbit α-eIF4E (catalog number 9742), rabbit α-PARP (catalog number 9542), α-SV40 large T antigen (catalog number 15729) all from Cell Signaling Technology (Danvers, MA) at a 1:1000 dilution, mouse α-β-actin (Sigma-Aldrich, catalog number A1978) at a 1:10,000 dilution and rabbit α-eIF4GI (kindly provided by Nahum Sonenberg, McGill University Montreal, QC, Canada) at a 1:2500 dilution. Preceding and following incubation with the appropriate horseradish peroxidase labeled secondary antibodies, the blots were washed three times for 5 minutes in TBST and detection was then performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest.