Figure 1: NM1 and Myo1c localize predominantly to the cytoplasm.

(A) Hela cells were fractionated into the cytoplasm (C) and nuclei (N), and the same amounts of proteins from each fraction were resolved by SDS-PAGE as shown on ponceau stained gel. The purity of each fraction was assessed using lamin A/C and GAPDH as markers. (B) Plasma membrane fragments were resolved by SDS-PAGE. ATPase and cadherin were used as markers of the plasma membrane fraction purity and as a loading control. Empty beads were used as negative control. NM1 or both NM1+Myo1c were detected by corresponding antibodies using western blotting. (C) Densitometric analysis of the immunoblotting shown in (A) shows that there is predominant localization of NM1 and Myo1C in the cytoplasm in comparison to the nucleus. (D) Densitometric analysis of the immunoblotting shown in (B) suggests that there is the same amount of NM1 as Myo1c at the plasma membrane and that KO of NM1 does not cause translocation of Myo1c to/from the plasma membrane. In both analyses, NM1 and Myo1c are detected by different antibodies; therefore, the signal of these antibodies was normalized to the known amount of purified NM1-Flag present within the same gel (not shown) and used for quantification. The experiment was repeated three times with consistent results.