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Fig. 8 | Molecular Brain

Fig. 8

From: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

Fig. 8

Interaction of full length or tail-less (ΔT) recombinant myo6 with Na+/K+-ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na+/K+-ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na+/K+-ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na+/K+-ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na+/K+-ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15: Figure S14

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