Hrp conjugated secondary antibody

A trichloroacetic acid (TCA) precipitation method was used to prepare cell lysates from WT and rpa14Δ strains. Briefly, 10 ml of log-phase cells were harvested and washed once with 1 ml of 20% TCA. The pellets were resuspended with 0.5 ml of 20% TCA and transferred to 1.5 ml microfuge tubes which already contained 0.5 ml of glass beads. The tubes were vortexed 4 times for 30 s and then allowed to settle for 5 min on the bench. The lysates were transferred to new microfuge tubes. Glass beads in the first tube were washed twice with 0.5 ml of 5% TCA, with each wash being added to the new microfuge tube. The lysates were precipitated at 3000 rpm (800 xg) for 10 min at 4°C. The pellets were resuspended in 200 μl of 1x SDS sample buffer and neutralized by adding 50 μl of 2M Tris base. The SDS samples were boiled for 5 min and 10 μl loaded onto a 9% SDS-polyacrylamide gel. Proteins were transferred to Immobilon-P membranes (Millipore) using a BioRad semi-dry transfer apparatus. Membranes were incubated with 1:3000 dilutions of α-tubulin monoclonal antibody (B-5-1-2, Sigma), polyclonal α-Fob1 antibody (yL-18, Santa Cruz) or polyclonal α-Sir2 antibody (YN-19, Santa Cruz). HRP-conjugated secondary antibodies from Promega were used at 1:5000 dilutions, and images developed with HyGLO (Denville Scientific).

Total protein was extracted from cultured cells and analyzed as previously described 27 (link), 28 (link). A total of 50 μg of protein extracts were loaded to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). After incubated with the primary antibodies overnight at 4 ℃ and then with HRP conjugated secondary antibodies (Promega, USA) for 1 h at room temperature, the membranes were subjected to the ECL chemiluminescence system (Pierce, USA). All of the experiments were repeated thrice. Primary antibodies used: CBX8 from Sigma Aldrich; Snail, Slug and Vimentin from BD Biosciences; E-cadherin and N-cadherin from Cell Signaling Technology; CBX6, Fibronectin and GAPDH from Santa Cruz.

For preparation of cell lysates for immunoblotting, the cells were collected and lysed by vortexing at 4°C in lysis buffer (20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM dithiothreitol, and 1/1000 protease 26 inhibitor cocktail (Nacalai Tesque). Lysates were cleared by centrifugation for 10 min at 13,000 rpm at 4°C, and the supernatants were collected for immunoblotting. SDS–PAGE was performed using 6–12% polyacrylamide gels, followed by transfer on Immobilon-P membrane (Millipore). The membranes were probed with the primary antibodies, followed by incubation with their respective HRP-conjugated secondary antibodies (Promega). Washes were performed in PBS containing 0.02% Tween. The signals were detected by a Chemi Doc XRS+ (Bio-Rad), and band intensities were calculated using the inbuilt Image Lab software (Bio-Rad). Unless otherwise specified, all immunoblotting analyses were repeated at least three times. The antibody against α-tubulin was used as a loading control.

Total protein extracts were prepared from mouse liver or human HCC specimens by lysis with a solution comprising 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, aprotinin (10 µg/ml), leupeptin (10 µg/ml), 1 mM PMSF, 400 µM Na₃VO₄, 400 µM EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The extracts (40 µg of protein) were fractionated by SDS-PAGE on a 10% gel, and the separated proteins were then transferred to a polyvinylidene difluoride membrane (IPVH00010; Millipore). The membrane was exposed to 3% dried skim milk and then incubated overnight at 4°C with primary antibodies to TfR1 (13–6800; Invitrogen), to Hmox1 (sc-10789; Santa Cruz Biotechnology), to Hsp90 (610419; BD Biosciences), to human IRP2 (sc-33682; Santa Cruz Biotechnology), or to mouse IRP2 (generated by K. Iwai, Kyoto University, Kyoto, Japan). Immune complexes were detected with HRP-conjugated secondary antibodies (Promega) and enhanced chemiluminescence reagents (Thermo Fisher Scientific; Nagahama et al., 2001 (link); Foster et al., 2003 (link); Hirano et al., 2013 (link)). For analysis of the human HCC specimens, the membrane was also stained with Coomassie Brilliant Blue as a loading control.