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Fig. S1. Comparison of flippase activity between ATP11C-a, ATP11C-b, and ATP11C-b(3ala)-expressing

cells. (A) Parental MDA-MB-231 cells (-) and MDA-MB- 231 cells stably expressing ATP11C-a, ATP11C-b, or

ATP11C-b(3ala) were washed with flippase assay buffer and incubated with NBD-labelled lipids (NBD-PS for 5

min or NBE-PE for 15 min) at 15°C. After extraction with fatty acid-free BSA, the residual fluorescence

intensity of the cells was measured by flow cytometry. Uptake of NBD-labelled lipids is shown relative to

parental cells (-). Graphs ± SD display the average of three independent experiments. Variance was assessed by

comparison of all pairs using a Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. (B)

MDA-MB- 231 cells stably expressing ATP11C-a, ATP11C-b, or ATP11C-b(3ala), and parental cells (-), were

lysed and subjected to immunoblotting using anti-H A, anti-ezrin, or anti-β-actin antibodies.

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Fig. S2. Localization of endogenous ezrin in cells expressing ATP11C-a, ATP11C-b, or ATP11C-b(3ala). (A)

Parental MDA-MB-231 cells (-) and MDA-MB-231 cells stably expressing (B) ATP11C-a, (C) ATP11C-b, or (D)

ATP11C-b(3ala) were fixed and immunostained using anti-ezrin alone (A) or anti-ezrin and anti-HA antibodies (BD) followed by incubation with Alexa555-conjugated anti-mouse secondary antibody and Alexa488-conjugated

phalloidin (A) or Alexa488-conjugated anti-mouse and Cy3-conjugated anti-rat secondary antibodies (B-D).

Scale bars, 20 μm. Scale bars in enlarged images, 5 μm. (E) Schematic illustration of localization of endogenous

ezrin and each ATP11C isoform and corresponding mutants. Orange, endogenous ezrin; light blue, ATP11C-a;

pink, ATP11C-b. (F) MDA-MB-231 cells and (G) BaF3 cells stably co-expressing HA-tagged ATP11C-b and

FLAG-tagged ezrin(WT) or ezrin(T567A) (TA) were fixed and stained for HA and FLAG followed by

incubation with Cy3-conjugated anti-rat and DyLight649-conjugated anti-mouse secondary antibodies and

Alexa488-conjugated phalloidin (F), or Alexa488-conjugated anti-rat and DyLight649-conjugated anti-mouse

secondary antibody and Alexa555-conjugated phalloidin (G). Scale bars, 20 μm (F) and 5 μm (G).

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Fig. S3. Estimation of polarized localization of ezrin(WT) and the phosphomimetic ezrin mutant. (A)

A representative line-scan profile from the uropod to the front of a BaF3 cell expressing ezrin(WT). The line scan was

performed using the ZEN software. (B) The ratio of the peak fluorescence intensity of the uropod to front (i/ii) of a BaF3

cell expressing ezrin(WT) or ezrin(T567D) was calculated and expressed as scatter plots, as described in the legend for

Figure 6D. Individual dots represent the ratios in individual cells. Graphs ± SD display the average ratio from all analyzed

cells, and ‘n’ is the total number of analyzed cells from two independent experiments. Variance was assessed by

comparison between ezrin(WT) and ezrin(T567D) using a Student’s t-test. *** p < 0.0001.

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Fig. S4. Polarized localization of the phosphomimetic radixin and moesin mutants. (A, B) MDA-MB-231 cells

and (C, D) BaF3 cells stably expressing C-terminally FLAG-tagged radixin(WT), radixin(T564D), moesin(WT), or

moesin(T558D) were fixed and stained for FLAG followed by incubation with Alexa555-conjugated anti-mouse

secondary antibody and Alexa488-conjugated phalloidin. Scale bars, 20 μm, 5 μm in enlarged images (A, B), and 10

μm (C, D).

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Fig. S5. Expression of exogenous ATP11C-a, ATP11C-b, and ATP11C-b(3ala) in Atp11c-knockout cells. (A)

Parental BaF3 cells (Con), Atp11c-knockout cells, and cells stably expressing CRISPR-resistant HA-tagged

ATP11C-a, ATP11C-b, or ATP11C-b(3ala) in the indicated Atp11c-knockout cell clone were lysed, and lysates

were subjected to SDS-PAGE and immunoblotting using anti-HA or anti-β-actin (as an internal control) antibodies.

(B) Cells in (A) were washed with flippase assay buffer and incubated with NBD-PS for 5 min, NBD-PE for 15

min, or NBD-PC for 15 min at 15°C. After extraction with fatty acid-free BSA, cellular fluorescence intensity was

measured by flow cytometry. Uptake of NBD-conjugated lipids is shown relative to parental cells (Con). Graphs ±

SD display the average of three independent experiments. A one-way ANOVA was performed to assess variance, and

comparisons to parental cells (Con) were made with Dunnett’s analysis. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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Fig. S6. MPAct probe recognizes membrane proximal F-actin (Bisaria et al., 2020)

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Fig. S7. Enrichment of MPAct at the uropod in BaF3 cells

Parental BaF3 cells (Con) and Atp11c-knockout cells stably expressing MPAct-mRuby3 were fixed and incubated with

Alexa488-conjugated phalloidin. Line-scan profiles of MPAct and phalloidin fluorescence from the uropod to the front of

these cells are shown. Different colors of lines represent different cells and ‘n’ is the total number of analyzed cells.

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