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. 2016 Nov 15;129(22):4252–4264. doi: 10.1242/jcs.194332

Fig. 1.

Fig. 1.

Tm1C is an interacting partner of Khc. (A) Schematic of the Tm1 locus. Untranslated regions are in gray and coding regions are depicted in orange. (B) Co-immunoprecipitation experiment in S2 cells between RFP–Khc and GFP (lanes 1, 3) or GFP–Tm1C (lanes 2, 4). The Khc construct did not contain the motor domain. The immunoprecipitation (IP) was performed using GFP-trap beads. The co-precipitating proteins and total fraction were analyzed by blotting using the indicated antibodies. (C) A co-immunoprecipitation experiment between RFP–Khc and GFP–Tm1C (lanes 1, 5), Tm1J (lanes 2, 6), Tm2A (lanes 3, 7) or Tm1A (lanes 4, 8). The immunoprecipitation was performed as in B and the co-precipitating proteins were detected by blotting using the indicated antibodies. The arrow indicates full-length GFP–Tm1C and the arrowhead denotes full-length GFP–Tm1J, Tm2A and Tm1A. Overexpression of GFP-tagged Tropomyosins in S2 cells often resulted in smaller faster-migrating bands (indicated by asterisks in this panel). We assume these bands represent breakdown products resulting from protein turnover. (D) A direct binding experiment using recombinant GST or GST–Tm1C produced in bacteria and RFP–Khc produced using in vitro transcription and translation. The binding reaction was run on a gel and analyzed by blotting using an anti-RFP antibody (top panel). The bottom panel is a GelCode-Blue-stained gel showing the amount of GST and GST–Tm1C used in the binding reaction. (E) The domain structure of Tm1A, Tm1J and Tm1C proteins is shown at the top of the figure. Numbers correspond to the amino acid positions for the various domains. The bottom part of the figure shows a co-immunoprecipitation experiment using RFP–Khc and either GFP (lanes 1, 5), full-length GFP–Tm1C (lanes 2, 6), an N-terminal GFP–Tm1C construct (lanes 3, 7) or a C-terminal GFP–Tm1C construct (lanes 4, 8). Immunoprecipitation was performed as in B. The arrow indicates full-length GFP–Tm1C, the arrowhead corresponds to N-terminally truncated GFP–Tm1C and the asterisk denotes C-terminally truncated GFP–Tm1C.