Figure 4. Cellular localization of Msi1.... | Rockefeller University Press

Figure 4.

$Figure 4. Cellular localization of Msi1. (A) Msi1 localizes to cytoplasmic foci. P19 cells treated with (right columns; 44°C for 30 min) or without (left columns) heat stress were stained with anti-Msi1 (green), and anti-hRAP55 (red, top) or anti-Dcp1a (red, bottom) antibodies, respectively. Nuclei were stained with TO-PRO-3 (blue) in the merged images. The white arrowheads and white arrow indicate PBs and SGs, respectively. Bars, 5 μm. (B) Msi1-positive granules were analyzed by two methods assessing the percent colocalization (1) or weighted colocalization coefficient (2) of their ratio to Dcp1a-, hRAP55-, PABP-, and eIF4G-containing granules. Msi1 mostly localized to SGs, but some was localized to PBs. (C) Association of Msi1 with heavy-sedimenting particles in an RNA-dependent manner. Each subcellular fraction with (lanes 4–6) or without (lanes 1–3) RNase A treatment after ultracentrifugation (top panels). S100, supernatants after ultracentrifugation; P100, pellets after ultracentrifugation. Total RNAs purified from S100 (lanes 2 and 5) and P100 (lanes 3 and 6) are shown (bottom panel). (D) P19 cells treated with (left) or without (right) heat stress were separated by 15–40% sucrose density gradients. Immunoblots of the gradient fractions were probed using antibodies against the indicated proteins (bottom panels).$

Cellular localization of Msi1. (A) Msi1 localizes to cytoplasmic foci. P19 cells treated with (right columns; 44°C for 30 min) or without (left columns) heat stress were stained with anti-Msi1 (green), and anti-hRAP55 (red, top) or anti-Dcp1a (red, bottom) antibodies, respectively. Nuclei were stained with TO-PRO-3 (blue) in the merged images. The white arrowheads and white arrow indicate PBs and SGs, respectively. Bars, 5 μm. (B) Msi1-positive granules were analyzed by two methods assessing the percent colocalization (1) or weighted colocalization coefficient (2) of their ratio to Dcp1a-, hRAP55-, PABP-, and eIF4G-containing granules. Msi1 mostly localized to SGs, but some was localized to PBs. (C) Association of Msi1 with heavy-sedimenting particles in an RNA-dependent manner. Each subcellular fraction with (lanes 4–6) or without (lanes 1–3) RNase A treatment after ultracentrifugation (top panels). S100, supernatants after ultracentrifugation; P100, pellets after ultracentrifugation. Total RNAs purified from S100 (lanes 2 and 5) and P100 (lanes 3 and 6) are shown (bottom panel). (D) P19 cells treated with (left) or without (right) heat stress were separated by 15–40% sucrose density gradients. Immunoblots of the gradient fractions were probed using antibodies against the indicated proteins (bottom panels).

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