Figure 7.
A single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention. (A) Immunostaining of Kapβ1 in cells expressing mCherry-CAS_T18D in the presence and absence of MEK1 inhibitor. Cells expressing mCherry-CAS_T18G, which has a neutral mutation at the same position is shown for comparison. Scale bar, 5 μm. (B) N/C ratios for mCherry-CAS_T18G and mCherry-CAS_T18D in the presence and absence of MEK1 inhibitor. The dashed line indicates N/C = 1. Box plots denote the median, first, and third quartiles. The whiskers represent the minimum and maximum values. Median values are indicated next to the box plots. N = 3 or 4. The data were tested for normality using a built-in function of GraphPad Prism software. P values were obtained using unpaired two-tailed t test. (C) Filamentous actin (F-actin) is stained using mEos2-Lifeact-7 peptide in cells expressing mCherry-CAS, mCherry-CAS_T18G, and mCherry-CAS_T18D. Only mCherry-CAS_T18D lacks retention in the nucleus and enriches with cytoplasmic micro-vesicles and invadopodia (indicated by arrows). Magnification of the bounded region (red) in Row 3 is shown in Row 4. Colocalization of F-actin and mCherryCAS_T18D gives a Pearson’s coefficient of 0.767. Scale bar, 5 µm. (D) The wound healing rate of mCherry-CAS_T18D cells (green) is the fastest overall. This is followed by mCherry-CAS (black) and mCherry-CAS_T18G (blue) cells. Non-transfected cells are shown to be slowest (red). Note: The slope of mCherry-CAS is omitted due to its similarity to mCherry-CAS_T18G. N = 3.

A single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention. (A) Immunostaining of Kapβ1 in cells expressing mCherry-CAS_T18D in the presence and absence of MEK1 inhibitor. Cells expressing mCherry-CAS_T18G, which has a neutral mutation at the same position is shown for comparison. Scale bar, 5 μm. (B) N/C ratios for mCherry-CAS_T18G and mCherry-CAS_T18D in the presence and absence of MEK1 inhibitor. The dashed line indicates N/C = 1. Box plots denote the median, first, and third quartiles. The whiskers represent the minimum and maximum values. Median values are indicated next to the box plots. N = 3 or 4. The data were tested for normality using a built-in function of GraphPad Prism software. P values were obtained using unpaired two-tailed t test. (C) Filamentous actin (F-actin) is stained using mEos2-Lifeact-7 peptide in cells expressing mCherry-CAS, mCherry-CAS_T18G, and mCherry-CAS_T18D. Only mCherry-CAS_T18D lacks retention in the nucleus and enriches with cytoplasmic micro-vesicles and invadopodia (indicated by arrows). Magnification of the bounded region (red) in Row 3 is shown in Row 4. Colocalization of F-actin and mCherryCAS_T18D gives a Pearson’s coefficient of 0.767. Scale bar, 5 µm. (D) The wound healing rate of mCherry-CAS_T18D cells (green) is the fastest overall. This is followed by mCherry-CAS (black) and mCherry-CAS_T18G (blue) cells. Non-transfected cells are shown to be slowest (red). Note: The slope of mCherry-CAS is omitted due to its similarity to mCherry-CAS_T18G. N = 3.

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