Figure 5.
NAP1 expression level is controlled by the UPS. (A and B) Representative Western blot and semiquantitative analysis of NAP1 normalized to GAPDH in G2, mitotic (M), and G1-asynchronous conditions in RPE-1 cells. RO-3306 was used for synchronization. Error bars indicate ± SEM; n = 3 independent experiments. (C) Relative NAP1 mRNA expression normalized β-actin in asynchronous and synchronized mitotic HeLa cells. Nocodazole was used for synchronization. Error bars indicate ± SD; n = 3 independent experiments. (D) Relative NAP1 mRNA expression normalized to β-actin in G2, mitotic, and G1-asynchronous RPE-1 cells. Error bars indicate ± SD; n = 3 independent experiments. Cells were synchronized at G2 using RO-3306. (E–G) Western blot analysis of NAP1 protein levels after cycloheximide treatment up to 6 h in HeLa (E), RPE-1 (F), and DLD-1 (G) cells. (H–J) Western blot analysis of NAP1 protein levels after 6 h of chloroquine treatment in HeLa (H), RPE-1 (I), and DLD-1 (J) cells. (K–M) Western blot analysis of NAP1 protein level after MG132 treatment followed by G2 release in HeLa (K), RPE-1 (L), and DLD-1 (M) cells. RO-3306 was used for synchronization. (N) Western blot analysis of NAP1 levels every 5 min after G2 release in RPE-1 cells. RO-3306 was used for synchronization. (O) Representative confocal images of FKBP12F36V-NAP1 DLD-1 cells (untreated) with immunocytochemical detection of HA (red). DAPI (blue) was used as a nuclear counterstain and α-tubulin for cytoskeleton staining (green). A dashed outline indicates the cell border. Scale bar = 10 μm. One dot = one independent experiment. Student’s t test or one-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ns = not significant. Source data are available for this figure: SourceData F5.

NAP1 expression level is controlled by the UPS. (A and B) Representative Western blot and semiquantitative analysis of NAP1 normalized to GAPDH in G2, mitotic (M), and G1-asynchronous conditions in RPE-1 cells. RO-3306 was used for synchronization. Error bars indicate ± SEM; n = 3 independent experiments. (C) Relative NAP1 mRNA expression normalized β-actin in asynchronous and synchronized mitotic HeLa cells. Nocodazole was used for synchronization. Error bars indicate ± SD; n = 3 independent experiments. (D) Relative NAP1 mRNA expression normalized to β-actin in G2, mitotic, and G1-asynchronous RPE-1 cells. Error bars indicate ± SD; n = 3 independent experiments. Cells were synchronized at G2 using RO-3306. (E–G) Western blot analysis of NAP1 protein levels after cycloheximide treatment up to 6 h in HeLa (E), RPE-1 (F), and DLD-1 (G) cells. (H–J) Western blot analysis of NAP1 protein levels after 6 h of chloroquine treatment in HeLa (H), RPE-1 (I), and DLD-1 (J) cells. (K–M) Western blot analysis of NAP1 protein level after MG132 treatment followed by G2 release in HeLa (K), RPE-1 (L), and DLD-1 (M) cells. RO-3306 was used for synchronization. (N) Western blot analysis of NAP1 levels every 5 min after G2 release in RPE-1 cells. RO-3306 was used for synchronization. (O) Representative confocal images of FKBP12F36V-NAP1 DLD-1 cells (untreated) with immunocytochemical detection of HA (red). DAPI (blue) was used as a nuclear counterstain and α-tubulin for cytoskeleton staining (green). A dashed outline indicates the cell border. Scale bar = 10 μm. One dot = one independent experiment. Student’s t test or one-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ns = not significant. Source data are available for this figure: SourceData F5.

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