Labworks image acquisition and analysis software

The NPC tissues and normal tissues were washed with PBS, added with cell lysates containing with protease inhibitors, shaken at 4°C for 5 min and centrifuged at 4°C for 10 min at the rate 37100 × g. The proteins were extracted from the supernatant by using Qproteome Mammalian Protein Prer kit (QIAGEN, GmbH, Germany). Proteins (50 μg) were obtained for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then moved to nitrocellulose membranes. Following blocking with skimmed milk and incubated overnight with the following primary antibody IL-6 (Cat# ab9324, 0.4 μg/ml, Abcam, Cambridge, MA, USA), JAK (Cat# ab205223, 1: 500, Abcam, Cambridge, MA, USA), p-JAK (Cat# ab32101, 1: 1000, Abcam, Cambridge, MA, USA), STAT3 (Cat# 119352, 1:600, Abcam, Cambridge, MA, USA), p-STAT3 (Cat# ab76315, 1:2000, Abcam, Cambridge, MA, USA) and CyclinD1 (Cat# ab134175, 1: 10000, Abcam, Cambridge, MA, USA). After washing with tris-buffered saline solution containing Tween 20 (TBST) 4 times (10 min every time), the membranes were incubated with IRDye™ 700DX-labeled IgG (LI-COR Bioscience, NE, USA) antibody (dilution 1:1000, Upstate, NY, USA) at room temperature for 1 h, washed by TBST 4 times and developed with the substrates. The data were analyzed by LabWorks Image Acquisition and Analysis Software (UVP, Inc., Upland, CA, USA) to obtain the relative protein concentration.

Western blotting was performed to determine NR5A2 protein expression. The total protein was extracted from the PDAC tissues and normal tissues. Proteins were quantitated by the Bradford method according to the manufacturer’s protocol (Bio-Rad, Hercules, CA, USA). Samples (50 μg protein) were resolved on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were probed with antibodies for NR5A2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All of the antibodies were purchased from Abcam company and were used at a dilution of 1:500 for anti-NR5A2 and 1:1000 for anti-GAPDH. The LabWorks image acquisition and analysis software (UVP, Upland, CA, USA) was used to quantify band intensities.

Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer in the presence of protease inhibitors. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies against p53 (DO-1) and β-actin were purchased from Abcam. Rabbit anti-human HPV16 E6 antibody (BS1719-R, 1:400) was purchased from BIOSS. Secondary antibody against goat anti-rabbit immunoglobulin G horseradish peroxidase conjugate was purchased from AJ. Proteins (30 μg) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Then, the membrane was blocked with 5% non-fat milk for 2 h and incubated with primary antibodies. Proteins were detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific). For immunoreactivity, the membrane was rinsed with TBS, incubated overnight at 4°C with a primary antibody, and then washed and incubated for 1 h with secondary antibody. Immunoreactivity was detected using an enhanced chemiluminescence substrate (Thermo Fisher Scientific). Lab Works image acquisition and analysis software (UVP, LLC) was used to quantify band intensities.

Cell protein lysates were separated using 10% sodium dodecyl sulfate polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes (Roche Diagnostics, Mannheim, Germany), and then detected using PTEN antibodies (ab107918), pAKT (sc-7985-R). Protein loading was measured using a mouse anti-GAPDH monoclonal antibody (mAbcam 8245). Lab Works Image Acquisition and Analysis Software (UVP, Upland, CA, USA) were adopted to quantify the band intensities.

The proteins were resolved on an SDS denaturing polyacrylamide gel and then transferred onto a nitrocellulose membrane. Antibody to PDCD4 or GAPDH was incubated with the membranes overnight at 4°C. The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein expression was assessed by enhanced chemiluminescence and exposure to chemiluminescent film. LabWorks™ Image Acquisition and Analysis Software (UVP, Upland, CA) were used to quantify the band intensities. All the antibodies were purchased from Abcam (Cambridge, MA).