Bca protein estimation assay

Protein from treated and untreated cell lysates was estimated using the BCA protein estimation assay (Thermo Scientific). For Western blotting, anti-HSP70 antibody (Enzo life sciences), and anti-Sp1 antibodies (Cell Signaling) were used to check for levels of different proteins in the lysates. Actin was used as loading control. Actin antibody was obtained from Cell Signaling Technology.

Cells were washed with ice cold PBS, and lysed with cell lysis buffer (Cell Signaling Technology, Beverly, MA). Protein concentrations were determined using the BCA protein estimation assay (Thermo Fisher Scientific). Equal amounts of protein were combined with 5× sample buffer (250mM Tris-HCl pH6.8, 10% SDS, 30% glycerol, 5% β-mecaptoethanol, and 0.02% bromophenol blue) at a 1:4 ratio and separated on a 4–20% Tris-glycine pre-cast gel (Thermo Fisher Scientific). Proteins were transferred to a 0.45μm nitrocellulose membrane before blocking with 5% milk in TBS-T (20mM Tris, 150mM NaCL, 0.01% Tween-20) for 60 mins, and then incubation with specific antibodies. The blots were incubated with a HRP-linked secondary antibody, resolved with the RPN2232 chemiluminescence reagent (GE Healthcare, Little Chalfont, UK) and visualized using X-ray film on the ImageQuant LAS500 (GE Healthcare).

Indicated cells were seeded in 10-cm plates with 50–60% confluence and incubated for 24 hours. For the protein stability assay, related cells were treated with cycloheximide (CHX) for 0, 2, 4, and 8 hours. Then the cells were harvested, pelleted by centrifugation, and then resuspended in RIPA buffer containing protease inhibitor and 1% Phenylmethanesulfonyl Fluoride (PMSF), and the protein concentration of the cell extract was calculated by the BCA protein estimation assay (Thermo Scientific). Equal amount of protein was loaded and separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore). The protein bands were probed with antibodies with proper dilution and incubated at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. The bands were visualized by enhanced chemiluminescence. Tubulin was used as loading control.
For the sub-cellular fractionation assay, proteins of cytoplasmic and nuclear proportion of shRCC1 or shScr cells were separated using “Nuclear and Cytoplasmic Protein Extraction kit” (Beyotime Biotechnology). Protein samples of nuclear and cytoplasmic proportions were further subjected to western blotting analysis as described. Lamin B1 was used as loading control for nuclear samples. Tubulin was used as loading control for cytoplasmic samples.

Pancreatic tumor tissue or cultured pancreatic cancer cells were lysed using RIPA buffer (Boston Bioproducts) and protein concentration was estimated using the BCA protein estimation assay (Thermo Scientific). Protein levels were detected using anti-HIF-1α antibody (Novus Biologicals) and anti-beta-Actin antibody (Santa Cruz).

To analyze protein expression, parts of mice pancreases were lysed using RIPA lysis buffer (Boston Bioproducts, MA, USA) containing protease and phosphatase inhibitors (Roche, Switzerland), and protein concentration was estimated using the BCA protein estimation assay (Thermo Scientific, MA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to the nitrocellulose membrane. Blots were probed with antibodies against pancreatic amylase (ab199132), alpha SMA (ab5694) and beta tubulin (ab179513) (Abcam, UK, 1:1000 dilution), after washing and re-probed with the respective secondary antibody (Abcam, UK, 1:10000 dilution). The bands were visualized using super signal West Dura Extended Duration Subject (34075- Thermo Fisher) in a Chemi-Doc (Bio-Rad).