Investigation of the loop sequence length in PiT family members. A The amino acid lengths of loop 6 (L6) are plotted for nine PiT family members (H. sapiens PiT2, H. sapiens PiT1, N. crassa Pho-4+, A. thaliana Pht2_1, E. coli PiTA, and putative phosphate permeases from D. melanogaster, C. elegans, T. brucei, and A. fulgidus). The L6 lengths are defined by the predicted TM domains in the protein sequences of the PiT family members; see alignment in and legend to Additional File 1 Figure A (AF 1 A). The maximum limit of a loop length (42 amino acids) estimated in Figure 4B is indicated on the figure. It illustrates that loop lengths at 1 to 42 amino acids define a loop sequence and loop lengths at 43 amino acids or higher defines a domain. B The numbers of amino acids in loop 1 (L1) to loop 9 (L9) in the protein sequences listed in the legend to A are shown. The loop lengths were defined by the sequences connecting the predicted TM domains in the protein sequences for the nine PiT family members; see alignment in and legend to Additional File 1 Figure A (AF 1 A). Data are the mean value of (n) numbers of loops counted ±SEM, see Additional File 2 for data. The stippled line indicates the maximum length for a loop sequence (L3) which is ~ 42 amino acids given with 95% confidence (38.6 ±3.4 amino acids ~ 35 to 42 amino acids). Note that the 95% confidence interval for L7 is 42.9 ±28.8 amino acids, illustrating that this loop length is subjected to high uncertainty because of an unusually long L7 in E. coli PiTA. The 95% confidence interval for L7 calculated when excluding L7 E. coli PiTA is 28.3 ±2.4 amino acids. The topology model indicates the positions of L1 to L9; stippled loops indicate the observed variable lengths of L6 (the large intracellular domain).