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. 2013 Aug 5;110(34):E3162–E3170. doi: 10.1073/pnas.1312531110

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Fig. 4. — Direct interactions of in vitro-synthesized full-length BIG1 and its C fragment with NMHC IIA, MYPT1, and PP1cδ. (A and B) BIG1, full-length and three fragments (BIG1-N, -S, and -C), with N-terminal HA tags and NMHC IIA (pCMV6-XL6-myosin IIA) were synthesized singly in wheat germ extract systems and incubated together as indicated before IP of NMHC IIA. Samples of in vitro-synthesized proteins before (5%, In) and from IP (50%) with antibodies against NMHC IIA were reacted with antibodies against (A) BIG1 or (B) HA or NMHC IIA. Arrow indicates BIG1-N, arrowhead BIG1-S, and asterisk BIG1-C. (C and D) Samples of in vitro-synthesized full-length BIG1 and c-myc-MYPT1 or c-myc-PP1cδ were mixed before IP with antibodies against c-myc and reaction of collected proteins (50%) with indicated antibodies. (E and F) In vitro-synthesized MYPT1 or PP1cδ and HA-tagged fragments of BIG1 were mixed as in B before IP of c-myc and reaction of collected proteins with antibodies against c-myc (MYPT1 or PP1cδ) or HA (BIG1).