Anti 6xhis antibody pa1 983b

100 μM Phos-tag acrylamide (Wako) within hand-cast 12% acrylamide gels was used for visualization of phosphosites by western blot. SDS-PAGE gels (4–15% acrylamide, Bio-Rad) and Phos-tag gels were transferred onto PVDF membranes. Anti-His westerns were performed using 1:2,500 diluted rabbit Anti-6xHis antibody (PA1–983B, Thermo Fisher Scientific) in 5% w/v milk in TBST for 1 h and 1:10,000 diluted donkey anti-rabbit HRP (711–035-152, Jackson ImmunoResearch) in 5% w/v milk in TBST for 1 h. HRP-conjugated anti-MBP monoclonal antibody (E80385, New England Biolabs) was used at a 1:500 dilution in 5% w/v milk in TBST for 1 h. Phospho-specific primary antibodies used in Supplementary Fig. 2 are listed in Supplementary Data 3 and were used in 1:1,000 dilutions in 5% w/v milk in TBST for 1 h. Protein bands were then visualized using Clarity ECL substrate (Bio-Rad) and an Amersham Imager 600 (GE Healthcare Life Sciences). All full (uncropped) western blots can be found in Supplementary Note 4–10.