Figure 1.
Nucleocytoplasmic transport rates are sensitive to levels of GTP. (A) Model of photoactivatable NCT reporter, LINuS. (B) Timeline of LINuS photoactivation and recovery, with representative IF images in BJ-5ta cells stably expressing LINuS during photoactivation and recovery. Yellow and blue boxes denote areas of nuclear and cytoplasmic measurement, respectively. (C) Representative graph of LINuS nuclear localization with trend lines, the slopes of which provide import or export rates. (D) Ratiometric images (405 nm/488 nm) of BJ-5ta cells expressing either GEVALNull or GEVAL30 treated with MPA. (E) Quantification of the GEVAL ratiometric signal (405 nm/488 nm) for each cell expressing GEVALNull (control n = 186, MPA n = 223) or GEVAL30 (control n = 175, MPA n = 264). Results are from two independent replicates. (F) Import and export rates of BJ-5ta cells expressing LINuS treated with MPA to deplete GTP (control n = 43, MPA n = 43), then rescued with guanosine supplementation (MPA + guanosine n = 44). Results are from two independent replicates. (G) Ratiometric images (405 nm/488 nm) of BJ-5ta cells expressing either GEVALNull or GEVAL30 treated with cycloheximide. (H) Quantification of cells from G (GEVALNull control n = 203, cycloheximide n = 232, GEVAL30 control n = 197, cycloheximide n = 198; three independent experiments). (I) Import and export rates of BJ-5ta cells expressing LINuS treated with cycloheximide (control n = 74, cycloheximide n = 82; two independent replicates). (J) Import and export rates of BJ-5ta cells depleted of ATP with sodium azide and 2-deoxyglucose (control n = 43, -ATP n = 43; two independent replicates). Significance calculated using unpaired t test (E and H–J), one-way ANOVA with Tukey’s post hoc (F). ns P > 0.05, P***<0.001, P****<0.0001. Scale bars 10 µm.

Nucleocytoplasmic transport rates are sensitive to levels of GTP. (A) Model of photoactivatable NCT reporter, LINuS. (B) Timeline of LINuS photoactivation and recovery, with representative IF images in BJ-5ta cells stably expressing LINuS during photoactivation and recovery. Yellow and blue boxes denote areas of nuclear and cytoplasmic measurement, respectively. (C) Representative graph of LINuS nuclear localization with trend lines, the slopes of which provide import or export rates. (D) Ratiometric images (405 nm/488 nm) of BJ-5ta cells expressing either GEVALNull or GEVAL30 treated with MPA. (E) Quantification of the GEVAL ratiometric signal (405 nm/488 nm) for each cell expressing GEVALNull (control n = 186, MPA n = 223) or GEVAL30 (control n = 175, MPA n = 264). Results are from two independent replicates. (F) Import and export rates of BJ-5ta cells expressing LINuS treated with MPA to deplete GTP (control n = 43, MPA n = 43), then rescued with guanosine supplementation (MPA + guanosine n = 44). Results are from two independent replicates. (G) Ratiometric images (405 nm/488 nm) of BJ-5ta cells expressing either GEVALNull or GEVAL30 treated with cycloheximide. (H) Quantification of cells from G (GEVALNull control n = 203, cycloheximide n = 232, GEVAL30 control n = 197, cycloheximide n = 198; three independent experiments). (I) Import and export rates of BJ-5ta cells expressing LINuS treated with cycloheximide (control n = 74, cycloheximide n = 82; two independent replicates). (J) Import and export rates of BJ-5ta cells depleted of ATP with sodium azide and 2-deoxyglucose (control n = 43, -ATP n = 43; two independent replicates). Significance calculated using unpaired t test (E and H–J), one-way ANOVA with Tukey’s post hoc (F). ns P > 0.05, P***<0.001, P****<0.0001. Scale bars 10 µm.

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