PHLPP1 splice variants differentially regulate AKT and PKCα signaling in hippocampal neurons: characterization of PHLPP proteins in the adult hippocampus
Article first published online: 28 SEP 2010
© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry
Journal of Neurochemistry
Special Issue: Introducing Preclinical Systematic Reviews
Volume 115, Issue 4, pages 941–955, November 2010
How to Cite
Jackson, T. C., Verrier, J. D., Semple-Rowland, S., Kumar, A. and Foster, T. C. (2010), PHLPP1 splice variants differentially regulate AKT and PKCα signaling in hippocampal neurons: characterization of PHLPP proteins in the adult hippocampus. Journal of Neurochemistry, 115: 941–955. doi: 10.1111/j.1471-4159.2010.06984.x
- Issue published online: 21 OCT 2010
- Article first published online: 28 SEP 2010
- Accepted manuscript online: 2 SEP 2010 02:23PM EST
- Received May 24, 2010; revised manuscript received/accepted August 25, 2010.
Figure S1. Validation of hippocampal cell culture. Isolated hippocampal neurons were grown on glass cover slides for 10 days, and subsequently stained for cell markers indicated above each panel. All images represent overlays using cell type specific antibodies (GREEN) and nuclei (BLUE). No oligodendrocytes or microglia were detected, however, a small percentage (~ 5–7%) of cells were astrocytes. Hippocampal neurons represent the majority of cell types present in 10 DIV cultures. The bottom two figures show two random fields overlaying astrocytes (RED) and neurons (GREEN) for comparison. More neuronal staining is evident compared to astrocyte staining.
Figure S2. Cayman PHLPP1 antibody detects PHLPP1α. Blot showing that the PHLPP1 antibody, (Cayman Chemicals) used in all immunofluorescence experiments, also detects PHLPP1α by western analysis. The Cayman antibody detects PHLPP1α in HEK293-FT cells over-expressing human PHLPP1α but not in cells over-expressing GFP only. GAPDH (loading control) did not differ between PHLPP1α or GFP over-expressing cells.
Figure S3. Enhanced image of astrocyte & neurons. From white box in Fig. 3 Panel (i). Image shows an astrocyte in green devoid of RED PHLPP1 staining. Astrocytic processes extend around several large neuronal nuclei that show perinuclear PHLPP1 (RED) staining.
Figure S4. PHLPP1 brain immunofluorescence, control and CA1 228-ms exposure. (a–c) Control images showing little to no staining in brains incubated with secondary only. (a) red channel 228-ms exposure and (b) green channel. (d–f) The original exposure time (228-ms exposure) of hippocampal sub-region CA1 in the adult rat brain showing intense PHLPP1 (RED) staining.
Figure S5. Neuronal PHLPP1β knockdown alters basal activation of AKT, PKC, and ERK kinases. Hippocampal neurons were grown for 10 days in vitro (10 DIV). At DIV 0 and 7 neurons were transduced with either a non-targeting (NT) shRNA or selective PHLPP1β targeting shRNA. At DIV 10, cells were serum starved for 2 h, washed twice in ice cold PBS, and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Activation of AKT, PKC, and ERK were analyzed by western analysis. (a) Representative blots (n = 3) show without IGF-1 stimulation after a 2-h starvation period, AKT and PKC phosphorylation is reduced and ERK activation is increased in PHLPP1β knockdown neurons. (b–i) Densitometry of proteins analyzed by western blot (n = 5 for NT-Controls; n = 4 for PHLPP1β knockdown neurons). The results indicate PHLPP1β knockdown alters the basal activation of AKT, PKC, and ERK kinases. Data are significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Figure S6. Effect of PHLPP1 knockdown to alter AKT, PKC, and ERK activation in HEK293-FT cells. HEK293-FT cells were transduced with lentivirus delivering either a non-targeting (NT) shRNA or human specific PHLPP1 shRNA. (a) Western blot showing changes in phosphorylated/total levels of AKT, PKC, and ERK kinases after knockdown. (b and c) Densitometry of western blots in panel (a) (n = 4) showing knockdown of PHLPP1 proteins induced a significant increase (p = 0.02211) in pPKCα657 levels but not in PKCα total levels. Only PKCα phosphorylation was altered by PHLPP1 knockdown, no significant change in AKT or ERK phosphorylation was observed.
Figure S7. Effect of PHLPP1α over-expression to alter AKT, PKC, and ERK activation in HEK293-FT cells. HEK293-FT cells were transiently transfected with a plasmid containing either GFP only (i.e. control cells) or the human cDNA for PHLPP1α. (a) Western blot showing changes in phosphorylated/total levels of AKT, PKC, and ERK kinases after over-expression of PHLPP1α. (b) Densitometry of western blots in panel (a) (n = 3) showing pAKT473 levels had a tendency (p = 0.0776) to be higher in PHLPP1α over-expressing HEK293-FT cells. (c) pERK phosphorylation was significantly increased in PHLPP1α over-expressing cells (p = 0.0003).
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