Imagej software

After incubated with various concentrations of oxymatrine or SB203580 (Sigma-Aldrich, St Louis, MO; 10 μM), cells were suspended in 100μl of lysis buffer. After separating on 10 % SDS-polyacrylamide gel electrophoresis, proteins were transferred onto PVDF membranes. The membranes were subsequently blocked in defatted milk to reducing non-specific binding at 37˚C for 1 h and then incubated with different antibodies (MMP-2, MMP-9, p38, p-p38 and β-actin) in TBST containing 5% defatted milk at 4˚C overnight. Then membranes were incubated with corresponding second anti-body for 1 h at room temperature. The bands were measured with an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) and exposed by autoradiography. The densitometric analysis was carried out with Image J software (GE Healthcare, Buckinghamshire, UK).

Following the desired treatments, CHO cells were washed and harvested on ice, and then lyzed with ice-cold protein extraction buffer (Beyotime, Nanjing, China). The protein concentration was determined using BCA protein assay kit (ComWin, Beijing, China). Equal amounts of protein were loaded into gel wells, separated by electrophoresis on SDS-polyacrylamide gels and transferred onto PVDF transfer membrane (Millipore, Bedford, MA). The transferred blots were blocked with blocking agents (5% w/v BSA and 0.05% Tween-20 in TBS) for 1 h at room temperature. Blots were then incubated in a certain proportion of specific primary antibodies: anti-GRP78 (ab25192 Rabbit monoclonal, Abcam), anti-ATF-6 (ab203119 Rabbit polyclonal, Abcam), anti-PERK (C33E10 Rabbit polyclonal, Abcam), anti-IRE1 (ab37073 Rabbit polyclonal, Abcam), anti-CHOP (ab11419 Mouse monoclonal, Abcam), anti-Beclin1 (ab55878 Rabbit polyclonal, Abcam), anti-LC3B (ab81785 Rabbit polyclonal, Abcam) (1:1000) overnight at 4°C. These membranes were rinsed 3 times for 10 min each with TBS-Tween (Sigma, St. Louis, MO, USA) and incubated with HRP conjugated secondary antibodies (1:5000) (CWBio, Beijing, China). After washing, protein bands were visualized by an enhanced chemiluminescence detection kit (ComWin, Beijing, China). Each band density was quantified using Image J software (GE Healthcare, Piscataway, New Jersey, USA).

1×10⁶ cervical cancer cells were suspended in 250 µl of lysis buffer (40mmol/l Tris-HCl, 1 mmol/l EDTA, 150 mmol/l KCl, 100 mmol/l NaVO3, 1% Triton X-100, 1 mmol/l PMSF, pH 7.5). The nuclear lysates were harvested with NucBuster Protein Extration Kit (Novagen, Germany) according to manufacturer’s instructions. The proteins (50 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were subsequently blocked in non-fat milk (5% in Tris-buffered saline with TWEEN-20, TBST) buffer at 37°C for 1 h to block non-specific binding and were then incubated overnight with antibodies against p38, p-p38, MMP2, MMP9, TIMP-1, TIMP-2, NF-κB-p50, p65/RelA, Histone H1 or β-actin in TBST containing 5% defatted milk at 4°C. Then second antibodies were incubated. The bands were detected with an enhanced chemiluminescence kit (Amersham, ECL Plus, Freiburg, Germany) and exposed by autoradiography. The densitometry analysis was performed using Image J software (GE Healthcare, Buckinghamshire, UK).