Ecl western blotting system

Cells or bacteria were lysed with ice-cold RIPA buffer (Genestar Biotechnology, Kaohsiung, Taiwan). Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Protein sample (30–40 µg) was separated via SDS-PAGE using a Hoefer mini VE system (Amersham Biosciences, Piscataway, NJ, USA). Proteins were transferred to Immobilon-E polyvinylidene fluoride membrane (Merck Millipore, Carrigtwohill, County Cork, Ireland) according to the manufacturer’s instructions. Following the transfer, the membrane was washed with Tris-Buffered Saline (TBS) and blocked for 1 h at 37°C with 5% fat-free milk in TBS and 0.1% of Tween-20 (TBST). Diluted primary antibodies were added and co-incubated with the membrane at 4°C overnight. Blots were then incubated with peroxidase-conjugated secondary antibodies horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG. Following removal of the secondary antibody, blots were washed with TBST and developed using the ECL-western blotting system (Advansta, San Jose, CA, USA). Immunoblot densities were quantified using the Hansor Luminescence Imaging System LIS02 (Han-Shuo Life Technology, Taichung, Taiwan).

Cells were lysed in RIPA lysis buffer (25 mM Tris, 150 mM NaCl, 0.5% NP-40, 0.5% deoxycholate (w/v) and 0.1% SDS (w/v), pH 7.4) supplemented with Roche EDTA-free protease inhibitor cocktail (one tablet for 50 mL) and halt phosphatase inhibitor cocktail (Thermo Fisher Scientific), 10 min on ice. After lysate clearing by centrifugation (16,000 g, 10 min, 4 °C), proteins were quantified with BCA (Thermo Fisher Scientific) using BSA as standard. Cell lysates were resolved by regular or Phos-Tag-containing (20 or 50 μM, WAKO Chemicals) SDS–PAGE. After electrophoresis, Phos-Tag-containing SDS–PAGE were soaked in transfer buffer with 10 mM EDTA for 3 × 10 min, rinsed 10 min in transfer buffer without EDTA and blotted to nitrocellulose membranes (Amersham, Glattbrugg, Switzerland). Membranes were blocked with 5% non-fat dry milk in TBST and probed with the indicated antibodies. Binding was detected with anti-mouse-HRP secondary antibodies (no.115-035-003) or anti-rabbit-HRP secondary antibodies (no.111-035-003), from Jackson Laboratories, using the ECL western blotting system (Advansta). When multiple antibodies were used, equal amounts of samples were loaded on multiple SDS–PAGE gels and western blots were sequentially probed with a maximum of two antibodies. Uncropped immunoblot figures are provided as source data file.