Gel doc 200

Plasmid samples were fractionated by electrophoresis in a 0.7% agarose gel and DNA molecules were revealed by staining with ethidium bromide at 0.5 μg mL^-1. The image of the gels was obtained with GelDoc 200 (BioRad) and the bands were quantitated with the Quantity One 4.5.2 software (BioRad). The DNA fragments were transferred to a nylon membrane Biodyne A (PALL Gelman Laboratory, AnnArbor, MI, United States) by 5 inches Hg of vacuum for 2 h using the Vacuum Blotter model 785 (Bio-Rad). Internal regions of gutF, gutR and gutB genes were amplified by PCR generating amplicons 1, 2, and 3, respectively, in reactions catalyzed by PHFP, and by using as substrate total plasmidic DNA preparation of P. parvulus 2.6 and the primer pairs shown in Table 2. Then, the amplicons were labeled with digoxigenin-dUTP by using the DIG high prime DNA labeling and detection starter kit II (Roche, Mannheim, Germany). Each DIG-labeled DNA probe (25 ng mL^-1) was used for hybridization at 45°C following the specifications of the kit’s supplier. The hybridization bands were revealed with the chemiluminescent substrate CSPD, and the signals were detected with the LAS-3000 imaging system (Fujifilm, Stamford, CT, United States).

Cells are lysed and extracted using RIPA lysis buffer. Then, SDS-PAGE is used for electrophoretic separation and transferred to the PVDF membrane. Subsequently, the membrane is blocked with 5% skimmed milk for 1 h. Then, the primary antibody anti-Ki-67, anti-cleaved-caspase3, anti-cyclin D1, anti-MMP-2, anti-MMP-9, anti-AKT, anti-p-AKT, anti-p-mTOR, anti-p-4E-BP1, anti-p-JAK2, anti-JAK2, anti-p-STAT3, anti-STAT3, and anti-GAPDH (Abcam) are incubated at 4°C overnight. Subsequently, the secondary antibody labeled with horseradish peroxidase (HRP) is incubated for 1 hour at room temperature. Finally, the protein bands are exposed and analyzed using an ECL reagent on Gel-Doc 200 (Bio-Rad). GAPDH is used as an internal control.

When observing the different concentrations of TP influence on TLR4, cell
suspension in the logarithmic phase was seeded in a 6-well plate (2 mL, 5 ×
10⁵ cells per mL), and different concentrations of TP (5, 10, 20,
and 40 μg/mL) were incubated for 24 h. TP (40 μg/mL) was chose to treat cells
for 0, 6, 12, and 24 h. Cells were also lysed with ice-cold lysis buffer (50-mM
Tris-HCl, pH 6.8, 100-mM 2-mercaptoethanol, 2% w/v sodium dodecyl sulfate, and
10% glycerol). Proteins were separated with 10% sodium dodecyl
sulfate–polyacrylamide electrophoresis and then transferred onto a
polyvinylidene difluoride (PVDF) membrane at 100 V for 1.5 h. Proteins were
treated with Tris-buffered saline and Tween which contained 5% non-fat dried
milk for 1 h. Blots were incubated with primary antibodies (Rabbit Anti-human
TLR4, 1:800, Cell Signaling) at 4°C overnight. This was followed by incubation
with secondary antibodies (HRP-labeled Goat Anti-Rabbit IgG (H + L), 1:2000,
Cell Signaling) at room temperature for 2 h. After being washed with PBST
(phosphate buffered saline + 1% Tween 20), blots were analyzed using Gel-Doc 200
(Bio-Rad). Bio-Rad (Hercules, CA, USA) GAPDH. GAPDH: glyceraldehyde phosphate
dehydrogenase was used as the internal reference.