Fig. 2. (A) Purified recombinant TbNRKC-6His protein (NRKC, black left arrowhead) phosphorylates /ß casein substrate in vitro and preferentially phosphorylates ß casein (lane 2 arrowhead). The negative control BSA (apparent molecular mass 67 kDa) is not phosphorylated (lane 3). Purified kinase-dead TbNRKC-6His protein (mNRKC) (red arrowhead) does not phosphorylate the /ß casein (lane 7) or BSA (lane 8). Controls: lane 1 TbNRKC alone, lane 4 /ß casein alone, lane 5 BSA alone, lane 6 mNRKC alone. (B) Corresponding Coomassie-Blue-stained SDS-PAGE as protein loading control. (C) Corresponding western blot with Isa-1 polyclonal antibody as recombinant protein loading control. Pre-stained (left) and non-prestained (right) molecular mass markers show a slight discrepancy in the relative migration in the gel. Note that the recombinant mNRKC protein (B, position indicated by red arrowhead) migrates at a lower molecular mass than the recombinant NRKC (B, position indicated by black arrowhead). Arrowheads indicate where mNRKC and NRKC would normally run on SDS-PAGE.