Hyperfilm mp film

The full-length CDSs of SAPK8, SAPK9 and SAPK10 were ligated into the pET28a vector; the CDS of the truncated TE was ligated into the pMAL-c2x vector (primers shown in Supplementary Table 2); the vectors were created with the In-Fusion Advantage PCR Cloning Kit (Takara). The proteins were expressed in E. coli and purified according to the user's manual. Phosphorylation assays were performed with 1 μg of recombinant fusion protein, MBP-TE_N195-His, MBP-TE_N195 (S77A)-His, MBP-TE_C155-His or MBP-TE_C155 (T457A)-His and 2 μg of His-SAPK protein in 30 μl of kinase buffer (40 mM Hepes, pH 7.5, 20 mM MgCl₂, 2 mM DTT, 10 μCi [³²P] γATP, 1 × proteinase inhibitor cocktail and 1 × phosphatase inhibitor cocktail), incubated at 30 °C for 1.5 h. The reaction was terminated by adding 6 μl sample buffer and heating at 100 °C for 5 min. After separation on a 10% SDS-PAGE gel, the gel was stained with Coomassie blue and imaged with a BIO-RAD Gel Doc XR+ imaging system. Then, the gel was exposed to GE Amersham hyperfilm MP film for detecting phosphorylated proteins.

Protein samples (20 µl) were separated on ready-made 10% Nu-PAGE polyacrylamide gels (Invitrogen) by SDS-PAGE and transferred to Hybond-P nitrocellulose (GE Life Science) by semi-dry blotting. Nitrocellulose membranes were prepared by treating with 5 ml Pierce MISER antibody extender (Fisher Scientific) for 10 min and washing 7 times with distilled water. They were then blocked in 5% milk in Tris-buffered saline containing 0.05% TWEEN-20 and 0.05% Triton X-100 (TBS-T-T) overnight at 4°C. The membranes were then incubated with primary antibodies diluted in 5% milk in TBS-T-T overnight at 4°C. They were then washed 5 times for 5 min in TBS-T-T, and incubated in anti-mouse IgG-HRP conjugate (Insight Biotechnology Ltd, Wembley, UK, 1∶5,000–1∶10,000) for 1 h at room temperature. After washing a further 5 times for 5 min in TBS-T-T, the membranes were incubated for 5 min in SuperSignal West Pico Chemiluminescent substrate (Fisher Scientific) before being exposed to Hyperfilm-MP film (GE Life Science, Little Chalfont, UK) for development. The blots were stripped in Pierce Stripper buffer (Fisher Scientific) for 10 min, washed 7 times in TBS-T-T, reblocked, and reprobed.