Research Paper Volume 13, Issue 12 pp 16165—16177

PTEN nuclear translocation enhances neuronal injury after hypoxia-ischemia via modulation of the nuclear factor-κB signaling pathway

PTEN nuclear translocation is an essential step in the modulation of ERK1/2. (A) Whole cell protein levels for total ERK and p-ERK1/2. There were no significant changes in total ERK protein levels but p-ERK1/2 levels increased significantly in GFP-PTENK13R neurons after OGD. (B) Quantification of whole cell ERK1/2 and p- ERK1/2 expression, normalized against GFP-PTENWT neurons. (C) The expression level of p-ERK1/2 in cytoplasmic and nuclear fractions. Western blot showed that p-ERK1/2 expression increased in both the nuclear and cytoplasmic fractions in GFP-PTENK13R neurons after OGD. (D) Quantification of nuclear ERK1/2 and p-ERK1/2 expression, normalized against GFP-PTENWT neurons. (E) Quantification of cytoplasmic ERK1/2 and p-ERK1/2 expression, normalized against GFP-PTENWT neurons. (F) Immunofluorescence staining of nuclear NF-κB. The nuclear translocation of NF-κB increased in GFP-PTEN K13R neurons after OGD. The arrow indicates nuclear-NF-κB-positive cells with red fluorescence. (G) Qualification of fluorescence intensity of nuclear NF-κB, normalized against GFP-PTENWT neurons. n = 5 for all columns and ** p WT neurons; K13R: GFP-PTENK13R neurons.

Figure 4. PTEN nuclear translocation is an essential step in the modulation of ERK1/2. (A) Whole cell protein levels for total ERK and p-ERK1/2. There were no significant changes in total ERK protein levels but p-ERK1/2 levels increased significantly in GFP-PTENK13R neurons after OGD. (B) Quantification of whole cell ERK1/2 and p- ERK1/2 expression, normalized against GFP-PTENWT neurons. (C) The expression level of p-ERK1/2 in cytoplasmic and nuclear fractions. Western blot showed that p-ERK1/2 expression increased in both the nuclear and cytoplasmic fractions in GFP-PTENK13R neurons after OGD. (D) Quantification of nuclear ERK1/2 and p-ERK1/2 expression, normalized against GFP-PTENWT neurons. (E) Quantification of cytoplasmic ERK1/2 and p-ERK1/2 expression, normalized against GFP-PTENWT neurons. (F) Immunofluorescence staining of nuclear NF-κB. The nuclear translocation of NF-κB increased in GFP-PTEN K13R neurons after OGD. The arrow indicates nuclear-NF-κB-positive cells with red fluorescence. (G) Qualification of fluorescence intensity of nuclear NF-κB, normalized against GFP-PTENWT neurons. n = 5 for all columns and ** p < 0.01, vs. WT. OGD: oxygen and glucose deprivation, WT: GFP-PTENWT neurons; K13R: GFP-PTENK13R neurons.