Ecl western blot system

Cell pellets were lyzed in RIPA-lysis buffer (20 mM TRIS–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP40, 1% sodium deoxycholate, 10 mM NaF, and 0.1% SDS), supplemented with EDTA-free protease inhibitor (Roche) for 15 min on ice, followed by 20,600g at 4°C for 15 min to separate from cellular debris. Protein concentration was then measured with BCA (Pierce) or Bradford (Bio-Rad), Laemmli buffer was added, and samples were separated by SDS–PAGE and transferred to nitrocellulose membranes (GE Healthcare). Then, the membranes were blocked for unspecific binding in 5% milk in TBS-T followed by incubation with indicated antibodies. After incubating in peroxidase-coupled secondary antibodies, the membranes were developed using the ECL Western blot system (Thermo Fisher Scientific).
Bands in Fig 4F were quantified using Image Lab 6.1 (Bio-Rad). First, the intensity of the DHPS-cDNA band and GAPDH band was quantified in each lane. A normalization factor was calculated by dividing the GAPDH band intensity of each lane with the GAPDH band intensity of the first lane (reference). The intensity of the DHPS-cDNA band was then multiplied by this normalization factor. Normalized DHPS-cDNA expression levels were then compared to normalized DHPS-cDNA expression levels in each lane and indicated as ratio (DHPS-cDNA/DHPS-cDNA⁽⁻⁾). All calculated intensities were background-subtracted.

The extracted total proteins were quantified by a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein samples were resolved by SDS-PAGE with 12% polyacrylamide gel, transferred onto a 0.2 μm PVDF membrane, blocked with PBST (PBS with 0.05% Tween-20 detergent) containing 5% skim milk powder for 1 h at RT, and then probed with thee appropriate primary antibodies for 2 h at RT. The membranes were washed 3 times with PBST, incubated with the appropriate HRP-conjugated secondary antibodies at a dilution of 1–10,000 for 1 h at RT, washed again 3 times with PBST, and then developed using the ECL Western blot system (Thermo Fisher Scientific, Waltham, MA, USA).