Journal: Scientific reports

Article Title: Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

doi: 10.1038/srep11529

Figure Lengend Snippet: Figure 1. Fe65 inhibits cell motility in breast cancer cells. (A), 5 × 104 control or Fe65 stable knockdown MDA-MB-231 cells were plated into trans-well inserts and 3% FBS was used in the medium at the bottom of 24-well plates to induce the migration and invasion. Cells were fixed and photographed 8 h after plating. (B), 5 × 104 control or Fe65 stable knockdown MDA-MB-361 cells were plated into trans-well inserts and 5% FBS was used in the medium at the bottom of 24-well plates to induce the migration and invasion. Cells were fixed and photographed 16 h after plating. Western blots show efficient Fe65 knockdown and β -actin blots are included as loading controls. Quantitative data are shown as bar graphs with each data point representing analyses in duplicate performed in parallel that were repeated three times (n = 6). Values are means ± SD. P-values were calculated with student’s t test (1 tailed distribution).

Article Snippet: For the stable knockdown in MDA-MB-231 and MDA-MB-361, cells were transfected with the retroviral pGFP-V-RS control or Fe65 shRNA plasmids from Origene (Rockville, MD) and 24 h post transfections, were continuously cultured in DMEM medium containing 1 μ g /mL puromycin.

Techniques: Control, Knockdown, Migration, Western Blot

Journal: Scientific reports

Article Title: Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

doi: 10.1038/srep11529

Figure Lengend Snippet: Figure 2. The effect of Fe65 on breast cancer cell motility is acetylation-sensitive. MDA-MB-231 cells were transfected with control or Fe65 siRNA. 40 h post transfections; cells were treated with DMSO, 1 μ M SAHA, 2 μ M tubastatin A (Tuba A) or 2 μ g/ml ACY1215 for 8 h. (A), Western blot analyses were performed to show efficient Fe65 knockdown and β -actin blot was included to show even loading. (B) and (C), Cell migration (B) and invasion (C) assays were performed as in Fig. 1A and photographed 16 h after plating. Quantitative data were presented as bar graphs and data statistics performed as described in Fig. 1.

Article Snippet: For the stable knockdown in MDA-MB-231 and MDA-MB-361, cells were transfected with the retroviral pGFP-V-RS control or Fe65 shRNA plasmids from Origene (Rockville, MD) and 24 h post transfections, were continuously cultured in DMEM medium containing 1 μ g /mL puromycin.

Techniques: Transfection, Control, Western Blot, Knockdown, Migration

Journal: Scientific reports

Article Title: Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

doi: 10.1038/srep11529

Figure Lengend Snippet: Figure 3. The ability of Fe65 to suppress cell motility depends on HDAC6 status. WT and HDAC6 KO MEFs were transfected with control or Fe65 siRNA for 48 h. (A), Western blots were performed to show the efficient knockdown of Fe65 and β -actin blot was included to show even loading. (B), 4 × 104 cells were plated into trans-well inserts and cells were fixed and photographed 16 h after plating. (C), Quantitative data were presented as bar graphs and data statistics performed as described in Fig. 1.

Article Snippet: For the stable knockdown in MDA-MB-231 and MDA-MB-361, cells were transfected with the retroviral pGFP-V-RS control or Fe65 shRNA plasmids from Origene (Rockville, MD) and 24 h post transfections, were continuously cultured in DMEM medium containing 1 μ g /mL puromycin.

Techniques: Transfection, Control, Western Blot, Knockdown

Journal: Scientific reports

Article Title: Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

doi: 10.1038/srep11529

Figure Lengend Snippet: Figure 4. Fe65 binds to cortactin to inhibit cell motility through its PTB1 domain. (A), 293T cells were transfected with 1.5 μ g Flag-cortactin and/or 1.5 μ g of Myc-Fe65. Cellular extracts were subjected to co-immunoprecipitation analyses with antibodies as indicated. (B), 293T cells were transfected with 1.5 μ g Flag-cortactin and 1.5 μ g of Myc-Fe65 or deletion constructs as indicated. Cellular extracts were subjected to co-immunoprecipitation analyses with antibodies as indicated. (C), 293T cells were transfected with Myc-Fe65 or deletion constructs as indicated. 5 × 105 transfected cells were plated into trans-well inserts 24 h post transfections. Cells were fixed and photographed 36 h after plating. (D) and (E), Quantitative data were presented as bar graphs and data statistics were performed as described in Fig. 1. a, p < 0.01; b, p > 0.05.

Article Snippet: For the stable knockdown in MDA-MB-231 and MDA-MB-361, cells were transfected with the retroviral pGFP-V-RS control or Fe65 shRNA plasmids from Origene (Rockville, MD) and 24 h post transfections, were continuously cultured in DMEM medium containing 1 μ g /mL puromycin.

Techniques: Transfection, Immunoprecipitation, Construct

Journal: Scientific reports

Article Title: Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

doi: 10.1038/srep11529

Figure Lengend Snippet: Figure 5. Tip60 binds to and acetylates cortactin through Fe65 to inhibit cell motility. (A), Inducible Fe65 stable knockdown clones of 293T cells were transfected with 1.5 μ g Flag-cortactin and 1.5 μ g of HA- Tip60 and treated with DMSO or 1 μ g/mL doxycycline as shown. Cellular extracts were subjected to co- immunoprecipitation analyses with antibodies as indicated. (B), Cells were transfected as in panel A and treated with DMSO, 300 ng/mL TSA and/or 1 μ g/mL doxycycline. Cellular extracts were subjected to co- immunoprecipitation analyses with antibodies as indicated. Acetyl-lysine (Ac-K) antibody is an antibody that specifically recognizes lysine-acetylated proteins. (C) and (D), 293T cells were transfected with HA-Tip60 together with control or Fe65 siRNA. 36 h post transfections, 5 × 105 transfected cells were plated into the inserts for trans-well assays. Western blots were performed to show Fe65 knockdown and β -actin blots were included as loading controls (panel C). Cells were fixed and photographed 24 h after plating (panel D). (E) and (F), Quantitative data were presented as bar graphs and data statistics performed as described in Fig. 1.

Article Snippet: For the stable knockdown in MDA-MB-231 and MDA-MB-361, cells were transfected with the retroviral pGFP-V-RS control or Fe65 shRNA plasmids from Origene (Rockville, MD) and 24 h post transfections, were continuously cultured in DMEM medium containing 1 μ g /mL puromycin.

Techniques: Knockdown, Clone Assay, Transfection, Immunoprecipitation, Control, Western Blot

Journal: Scientific reports

Article Title: Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

doi: 10.1038/srep11529

Figure Lengend Snippet: Figure 6. Fe65 inhibits invadopodia formation in breast cancer cells. (A), MDA-MB-231 cells stably expressing control or Fe65 shRNA were plated onto gelatin-coated glass coverslips and stained for cortactin (green), F-actin (phalloidin, red) and DAPI (blue). Representative immunofluorescence images were included to show invadopodia underneath the cells as orange dots in the merged panels. Quantitative data were presented as bar graphs to show the difference between control and Fe65 knockdown cells. Each data point represents the counting of 30 cells. Values are means ± SD. Statistical analyses were performed with student’s t test (1 tailed distribution). (B), A model depicting our current understanding how Fe65 suppresses breast cancer migration and invasion through the recruitment of Tip60 that opposes the activity of HDAC6 on cortactin acetylation.

Article Snippet: For the stable knockdown in MDA-MB-231 and MDA-MB-361, cells were transfected with the retroviral pGFP-V-RS control or Fe65 shRNA plasmids from Origene (Rockville, MD) and 24 h post transfections, were continuously cultured in DMEM medium containing 1 μ g /mL puromycin.

Techniques: Stable Transfection, Expressing, Control, shRNA, Staining, Immunofluorescence, Knockdown, Migration, Activity Assay

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Quantification of protein interaction in living cells by two-photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed-through.

doi: 10.1002/cyto.a.21150

Figure Lengend Snippet: Figure 4. Intermolecular FRET. A: Schematic representation of APP and Fe65 within the cellular membrane context. The APP and Fe65 are tagged at the C-terminal end respectively by the YFP and the mCherry. B: Fluorescence intensity image (526—535 nm region) of HEK-293 expressing APP-YFP excited at 900 nm and emission spectrum profile from the region of interest (ROI). C: Fluorescence intensity image (606—619 nm region) of HEK-293 expressing Fe65-mCherry excited at 900 nm and emission spectrum profile from the region of interest (ROI) of Fe65-mCherry excited at 900 (solid line) and at 940 nm (dashed line). D: Fluorescence intensity image (526—535 nm 1 606—619 nm) of HEK-293 cell co-expressing excited at 900 nm and mission spectrum profile from ROI. E: FRET efficiency pseudo-color images of cells co-expressing APP-YFP and Fe65-mCherry at different ratio. F: Correlation plot between FRET efficiency and ratio of Fe65-mCherry and APP-YFP. G: When using classical FRET pair, FRET signal is found to be contaminated by direct emission from the acceptor (Fa), accep- tor as well as from the donor (Fd) upon optimal excitation of the donor. H: In the case of FRET pair free from acceptor bleed-through, the FRET signal is can be separated from donor by spectral unmixing. As a result, the FRET and Fd were the only parameters to be taken into account resulting in the simplification of the FRET quantification. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The human amyloid precursor protein (APP; OriGene, SC111589) and Fe65 cDNAs (OriGene, SC111588) were subcloned into mammalian expression vectors pEYFPN1 and pmCherry-N1 (Clontech, BD Biosciences).

Techniques: Membrane, Fluorescence, Expressing

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 3. FE65-Beclin 1 interaction is essential to facilitate Beclin 1-mediated autophagy. (A) A GFP-LC3 cleavage assay was performed in WT HEK293 and FE65 KO cells, with and without CQ treatment. The knockout of FE65 decreased the level of free GFP from GFP-LC3 cleavage, and the overexpression of Beclin 1 failed to potentiate GFP-LC3 cleavage in FE65 KO cells. The bar chart shows the level of GFP; error bars represent standard deviation (SD), *** p < 0.001. (B) FE65, but not FE65∆Ct, increased endogenous LC3 lipidation in stably expressing cells, with and without Baf A1 treatment. The bar chart represents the level of LC3-II; error bars are shown as SD, *** p < 0.001, ** p < 0.01. (C) FE65-stably transfected cells showed a decrease in the p62/Beclin 1 ratio with and without Baf A1 treatment, whereas FE65∆Ct did not exhibit this decrease. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. *** p < 0.001, ** p < 0.01, * p < 0.05. (D,E) FE65 and FE65∆Ct were transfected in FE65 KO cells to assess autophagic activity. (D) Only FE65 was able to rescue the lipidation of LC3, regardless of CQ treatment. The bar chart illustrates the level of LC3-II; error bars are shown as SD, ** p < 0.01, * p < 0.05. (E) FE65 transfection in FE65 KO cells led to a significant decrease in the p62/Beclin 1 ratio, both with and without CQ treatment, while transfection with FE65∆Ct attenuated this effect. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. ** p < 0.01, * p < 0.05. (F) FE65 and FE65∆Ct were transfected into GFP-LC3 stably expressing cells, with and without Baf A1 treatment.

Article Snippet: The levels of LC3, p62, FE65, Beclin 1, and tubulin were detected by anti-LC3 antibody (ProteinTech, San Biology 2025, 14, 97 5 of 20 Diego, CA, USA), anti-p62 antibody (ProteinTech), anti-FE65 antibody (E20, Santa Cruz, Dallas, TX, USA), anti-Beclin 1 antibody (rat), and anti-tubulin (DM1A, Santa Cruz).

Techniques: Cleavage Assay, Knock-Out, Over Expression, Standard Deviation, Stable Transfection, Expressing, Transfection, Activity Assay

Journal: Biological Research

Article Title: Aβ promotes CD38 expression in senescent microglia in Alzheimer’s disease

doi: 10.1186/s40659-022-00379-1

Figure Lengend Snippet: Aβ1-40 induced energy metabolism disorder and mitochondrial dysfunction in BV2 cells. Scale bar: 50 μm. A Morphological changes of BV2 cells treated with Aβ1-40 (1 μM). B SA-β-Gal positive stained area. C Representative images of BV2 cells immunolabeled for P16 (red) and P21 (red). Nuclear staining (DAPI) is shown in blue. D Cell viability of BV2 cells E ATP level of BV2 cells. F NAD + level of BV2 cells. G NADH level of BV2 cells. H NAD + /NADH ratio of BV2 cells. I ROS level of BV2 cells. J MMP level of BV2 cells. K CD38 enzymatic activity of BV2 cells. L P16, P21, and CD38 protein expression level of BV2 cells. All data are expressed as the mean ± standard error. **P < 0.01 vs. control group

Article Snippet: Mouse microglia BV2 cells were purchased from the Cell Bank of Chinese Academy of Sciences; Dulbecco’s modified Eagle medium (12100046), fetal bovine serum (10099141C), and penicillin/streptomycin (10378016) were purchased from Gibco; Aβ protein fragment 1–40 (A1075) was purchased from Sigma-Aldrich; Lipofectamine 2000 Transfection Reagent (ab51243) was purchased from Abcam; CD38 siRNA (AM160089) was purchased from Invitrogen; CD38 CRISPR activating plasmid (sc-419548), CD38 antibody (SC374650), and M-IgGκ BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; anti-rabbit IgG HRP-linked antibody (5490S) and β-actin mouse monoclonal antibody (3700) were purchased from Cell Signaling Technology; P21 rabbit antibody (AF5252), P16 rabbit antibody (AF1069), Iba1 rabbit antibody (AF7143), Cell Proliferation and Cytotoxicity Assay Kit (C0009), Enhanced ATP Assay Kit (S0027), Reactive Oxygen Species Assay Kit (S0033S), Mitochondrial Membrane Potential Assay Kit (C2006), and Mouse amyloid beta peptide 1–42 ELISA Kit (MU30114) were purchased from Bioswamp; Mouse TNF-α ELISA Kit (PT512), Mouse IL-6 ELISA Kit (PT312), Mouse IL-1β ELISA Kit (PT512), Senescence β-Galactosidase Staining Kit (C0602), Alexa Fluor 488 goat anti-rabbit IgG (a0423), DAPI-staining solution (C1006), phosphate buffered saline (C0221A) and bovine serum albumin (ST025) were purchased from Shanghai Beyotime Biotechnology; and NAD/NADH Quantitation Colorimetric Kit (K337) was purchased from Biovision.

Techniques: Staining, Immunolabeling, Activity Assay, Expressing, Control

Journal: Biological Research

Article Title: Aβ promotes CD38 expression in senescent microglia in Alzheimer’s disease

doi: 10.1186/s40659-022-00379-1

Figure Lengend Snippet: CD38 knockout improves energy dysmetabolism and reduces the expression of inflammatory factors in Aβ1-40 injured BV2 cells. Scale bar: 50 μm. A Representative image of BV2 microglial cells exposed to Aβ1-40 (1 μM), and overexpressed or knockdown CD38 immunolabeled for CD38 (green). Nuclear staining (DAPI) is shown in blue. B CD38 protein expression level of BV2 cells. C NAD + level of BV2 cells. D NADH level of BV2 cells. E NAD + /NADH ratio of BV2 cells. F ATP level of BV2 cells. G ROS level of BV2 cells. H MMP level of BV2 cells. I IL-1β level of BV2 cells. J IL-6 level of BV2 cells. K TNF-α level of BV2 cells. All data are expressed as the mean ± standard error. **P < 0.01 vs. control group; ## P < 0.01 vs. Aβ1–40 group; △△ P < 0.01

Article Snippet: Mouse microglia BV2 cells were purchased from the Cell Bank of Chinese Academy of Sciences; Dulbecco’s modified Eagle medium (12100046), fetal bovine serum (10099141C), and penicillin/streptomycin (10378016) were purchased from Gibco; Aβ protein fragment 1–40 (A1075) was purchased from Sigma-Aldrich; Lipofectamine 2000 Transfection Reagent (ab51243) was purchased from Abcam; CD38 siRNA (AM160089) was purchased from Invitrogen; CD38 CRISPR activating plasmid (sc-419548), CD38 antibody (SC374650), and M-IgGκ BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; anti-rabbit IgG HRP-linked antibody (5490S) and β-actin mouse monoclonal antibody (3700) were purchased from Cell Signaling Technology; P21 rabbit antibody (AF5252), P16 rabbit antibody (AF1069), Iba1 rabbit antibody (AF7143), Cell Proliferation and Cytotoxicity Assay Kit (C0009), Enhanced ATP Assay Kit (S0027), Reactive Oxygen Species Assay Kit (S0033S), Mitochondrial Membrane Potential Assay Kit (C2006), and Mouse amyloid beta peptide 1–42 ELISA Kit (MU30114) were purchased from Bioswamp; Mouse TNF-α ELISA Kit (PT512), Mouse IL-6 ELISA Kit (PT312), Mouse IL-1β ELISA Kit (PT512), Senescence β-Galactosidase Staining Kit (C0602), Alexa Fluor 488 goat anti-rabbit IgG (a0423), DAPI-staining solution (C1006), phosphate buffered saline (C0221A) and bovine serum albumin (ST025) were purchased from Shanghai Beyotime Biotechnology; and NAD/NADH Quantitation Colorimetric Kit (K337) was purchased from Biovision.

Techniques: Knock-Out, Expressing, Knockdown, Immunolabeling, Staining, Control

Journal: Biological Research

Article Title: Aβ promotes CD38 expression in senescent microglia in Alzheimer’s disease

doi: 10.1186/s40659-022-00379-1

Figure Lengend Snippet: Inhibition of CD38 expression improves energy metabolism disorder in APP/PS1 mice and reduces the neuroinflammatory response. Scale bar: 50 μm. A ATP level of the hippocampus. B ATP level of the cortex. C NAD + level of the hippocampus. D NADH level of the hippocampus. E NAD + /NADH ratio of the hippocampus. F NAD + level of the cortex. G NADH level of the cortex. H NAD + /NADH ratio of the cortex. I MMP of the hippocampus. J MMP of the cortex. K ROS level of the hippocampus. L ROS level of the cortex. M IL-1β level of the serum. N TNF-α level of the serum. O IL-6 level of the serum. P IL-1β level of the hippocampus. Q TNF-α level of the hippocampus. R IL-6 level of the hippocampus. S IL-6 level of the cortex. T TNF-α level of the cortex. U Representative images of Iba1 + and senile plaque immunolabeling in the hippocampus and cortex. *P < 0.05 and **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. model group; △ P < 0.05 and △△ P < 0.01

Article Snippet: Mouse microglia BV2 cells were purchased from the Cell Bank of Chinese Academy of Sciences; Dulbecco’s modified Eagle medium (12100046), fetal bovine serum (10099141C), and penicillin/streptomycin (10378016) were purchased from Gibco; Aβ protein fragment 1–40 (A1075) was purchased from Sigma-Aldrich; Lipofectamine 2000 Transfection Reagent (ab51243) was purchased from Abcam; CD38 siRNA (AM160089) was purchased from Invitrogen; CD38 CRISPR activating plasmid (sc-419548), CD38 antibody (SC374650), and M-IgGκ BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; anti-rabbit IgG HRP-linked antibody (5490S) and β-actin mouse monoclonal antibody (3700) were purchased from Cell Signaling Technology; P21 rabbit antibody (AF5252), P16 rabbit antibody (AF1069), Iba1 rabbit antibody (AF7143), Cell Proliferation and Cytotoxicity Assay Kit (C0009), Enhanced ATP Assay Kit (S0027), Reactive Oxygen Species Assay Kit (S0033S), Mitochondrial Membrane Potential Assay Kit (C2006), and Mouse amyloid beta peptide 1–42 ELISA Kit (MU30114) were purchased from Bioswamp; Mouse TNF-α ELISA Kit (PT512), Mouse IL-6 ELISA Kit (PT312), Mouse IL-1β ELISA Kit (PT512), Senescence β-Galactosidase Staining Kit (C0602), Alexa Fluor 488 goat anti-rabbit IgG (a0423), DAPI-staining solution (C1006), phosphate buffered saline (C0221A) and bovine serum albumin (ST025) were purchased from Shanghai Beyotime Biotechnology; and NAD/NADH Quantitation Colorimetric Kit (K337) was purchased from Biovision.

Techniques: Inhibition, Expressing, Immunolabeling, Control

Journal: Biological Research

Article Title: Aβ promotes CD38 expression in senescent microglia in Alzheimer’s disease

doi: 10.1186/s40659-022-00379-1

Figure Lengend Snippet: Inhibition of CD38 expression improves the cognitive learning ability of APP/PS1 mice. A Escape latency. B Total time. C Total distance. D Platform crossings. E Swimming trajectory. F New object recognition index. G New object recognition trajectory. H Aβ1-42 concentration of the hippocampus. I Aβ1-42 concentration of the cortex. J Aβ1-42 concentration of the serum. *P < 0.05 and **P < 0.01 vs. control group; # P < 0.05 and ## P < 0.01 vs. model group; △ P < 0.05 and △△ P < 0.01

Article Snippet: Mouse microglia BV2 cells were purchased from the Cell Bank of Chinese Academy of Sciences; Dulbecco’s modified Eagle medium (12100046), fetal bovine serum (10099141C), and penicillin/streptomycin (10378016) were purchased from Gibco; Aβ protein fragment 1–40 (A1075) was purchased from Sigma-Aldrich; Lipofectamine 2000 Transfection Reagent (ab51243) was purchased from Abcam; CD38 siRNA (AM160089) was purchased from Invitrogen; CD38 CRISPR activating plasmid (sc-419548), CD38 antibody (SC374650), and M-IgGκ BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; anti-rabbit IgG HRP-linked antibody (5490S) and β-actin mouse monoclonal antibody (3700) were purchased from Cell Signaling Technology; P21 rabbit antibody (AF5252), P16 rabbit antibody (AF1069), Iba1 rabbit antibody (AF7143), Cell Proliferation and Cytotoxicity Assay Kit (C0009), Enhanced ATP Assay Kit (S0027), Reactive Oxygen Species Assay Kit (S0033S), Mitochondrial Membrane Potential Assay Kit (C2006), and Mouse amyloid beta peptide 1–42 ELISA Kit (MU30114) were purchased from Bioswamp; Mouse TNF-α ELISA Kit (PT512), Mouse IL-6 ELISA Kit (PT312), Mouse IL-1β ELISA Kit (PT512), Senescence β-Galactosidase Staining Kit (C0602), Alexa Fluor 488 goat anti-rabbit IgG (a0423), DAPI-staining solution (C1006), phosphate buffered saline (C0221A) and bovine serum albumin (ST025) were purchased from Shanghai Beyotime Biotechnology; and NAD/NADH Quantitation Colorimetric Kit (K337) was purchased from Biovision.

Techniques: Inhibition, Expressing, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Phosphorylation of FE65 at threonine 579 by GSK3β stimulates amyloid precursor protein processing

doi: 10.1038/s41598-017-12334-2

Figure Lengend Snippet: GSK3β phosphorylates FE65 T579 ( A ) Alignment of FE65 PTB2 sequences from various species. T597 is bolded (reference to human). Secondary structures of human FE65 PTB2 are indicated according to , . The position of β ct is also shown. ( B ) FE65 and FE65 T579A were transfected to HEK293 cells together with or without GSK3β. Cell lysates were resolved on 8% (top panel) and 12% (second panel) SDS-PAGE gels for determination of FE65 migration patterns and total FE65 levels, respectively. Anti-FE65 E20 antibody was used to detect FE65. Expression of HA-tagged GSK3β was confirmed by immunoblotting using anti-HA 12CA5 antibody. α-tubulin was detected by anti-α-tubulin DM1A antibody as loading control. UT, untransfected. The relative amount of phosphorylated-FE65 was analyzed by densitometry (LI-COR® Image Studio™ Software). n = 4. *P < 0.001. Results are means ± S.D. ( C ) Bacterially expressed His-tagged FE65 PTB2 (531–666) wild-type/T579A were incubated with HA-tagged GSK3β immunoprecipitated from transfected cell lysate together with [γ- 32 P]-ATP for an hour at 30 °C. Arrow denotes the specific phosphorylated band in lane 4. Lower panel shows the Coomassie Blue staining. RM: reaction mixture only without substrate. WT, His-tagged FE65 PTB2 (531–666) wild-type. T579A, His-tagged FE65 PTB2 (531–666) T579A mutant. ( D ) FE65 and FE65 T579A were transfected to HEK293 cells together with or without GSK3β. T579 phosphorylated FE65 was detected by pT579 FE65 phospho-specific antibody. UT indicates untransfected. FE65, GSK3β and α-tubulin were detected by immunoblotting. The relative amount of p579 FE65 level was analyzed by densitometry. n = 6. *P < 0.001. Results are means ± S.D. ( E ) Endogenous FE65 was immunoprecipitated from adult rat brain lysate by using anti-FE65 E20. FE65 in the brain lysate and immunoprecipitate were revealed by immunoblotting. T579 phosphorylated FE65 was detected with pT579 FE65 antibody. Arrow denotes T579 phosphorylated FE65.

Article Snippet: The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579.

Techniques: Transfection, SDS Page, Migration, Expressing, Western Blot, Control, Software, Incubation, Immunoprecipitation, Staining, Mutagenesis

Journal: Scientific Reports

Article Title: Phosphorylation of FE65 at threonine 579 by GSK3β stimulates amyloid precursor protein processing

doi: 10.1038/s41598-017-12334-2

Figure Lengend Snippet: GSK3β enhances FE65-mediated APP processing. ( A ) HEK293 cells were co-transfected with GAL4-APP, pFR-Luc and phRL-TK together with empty vector (EV), FE65 or FE65 + GSK3β. GSK3β potentiates the stimulatory effect of FE65 on APP-GAL4 cleavage. n = 5. *P < 0.001. Results are means ± S.D. ( B ) HEK293 cells were co-transfected with APP + FE65 with or without GSK3β. Levels of secreted Aβ1–40 (top graph) and 1–42 (bottom graph) were measured by corresponding Aβ ELISA kits. Overexpression of GSK3β enhances the effect of FE65 on both Aβ1–40 and 1–42 liberation. n = 5. *P < 0.001. Results are means ± S.D.

Article Snippet: The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: Scientific Reports

Article Title: Phosphorylation of FE65 at threonine 579 by GSK3β stimulates amyloid precursor protein processing

doi: 10.1038/s41598-017-12334-2

Figure Lengend Snippet: FE65 T579 phosphorylation potentiates FE65-mediated APP processing. ( A ) HEK293 cells were co-transfected with GAL4-APP, pFR-Luc and phRL-TK together with EV, FE65, FE65 T579A or FE65 T579E. Phosphomimetic mutation of FE65 T579 further enhances the effect of FE65 on APP-GAL4 cleavage. n = 5. *P < 0.001. Results are means ± S.D. The expression of FE65 and the mutants were determined by immunoblotting. ( B ) Cells were transfected with APP + BACE1 + FE65/T579A/T579E. Transfected cell lysates were resolved on 16% Tris-Tricine gel, and immunoblotted for APP CTFs. Arrows denote CTF-α, -β or -β’. UT: untransfected. Two exposures of CTF blot were shown ( C ) Cells were transfected with APP together with EV, FE65, FE65 T579A or FE65 T579E. Levels of secreted Aβ1–40 (top graph) and 1–42 (bottom graph) were measured by corresponding Aβ ELISA kits. FE65 T579E phosphomimetic mutation potentiates FE65-mediated Aβ liberation. n = 5. *P < 0.001. Results are means ± S.D.

Article Snippet: The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Phosphorylation of FE65 at threonine 579 by GSK3β stimulates amyloid precursor protein processing

doi: 10.1038/s41598-017-12334-2

Figure Lengend Snippet: FE65 T579 phosphorylation enhances FE65/APP interaction but suppresses FE65-PTB2 dimerization. ( A ) Cells were transfected with GST + FE65 PTB2 T579A or T579E and GST-FE65 PTB2 + GFP-FE65 PTB2 T579A or T579E. GST and GST-FE65 PTB2 were pulled down from the cell lysates. Levels of GST or GST-FE65 PTB2 and GFP-FE65 PTB2 present in cell lysates and pull-downs were determined with anti-GST and anti-GFP antibodies respectively. The relative amounts of GFP-PTB2 in the immunoprecipitates were analyzed by densitometry. n = 3. *P < 0.001. Results are means ± S.D. UT, untransfected. CHO cells were transfected with ( B ) APP and APP + FE65 T579A or T579E ( C ) APP + FE65 or APP + FE65 + GSK3β. FE65 was immunoprecipiated by anti-myc 9B11 antibody to its myc-tag. Levels of APP, FE65 and GSK3β present in cell lysates and immunocomplex were determined using anti-APP, anti-FE65 and anti-HA antibodies respectively. The relative amounts of APP level in the immunoprecipitates were analyzed by densitometry. n = 3. *P < 0.001. Results are means ± S.D. For ( B ) and ( C ), 40% of cell lysate was used for each immunoprecipitation, and + and - denotes the presence or absence of anti-myc antibody in the immunoprecipitation. 5% of lysates were loaded for both ( B ) and ( C ). UT, untransfected. IPs, immunoprecipitations. ( D ) Immunofluorescence staining of COS-7 cells with FE65 T579A, FE65 T579E, APP + FE65 T579A and APP + FE65 T579E. Nucleus were stained by DAPI. Scale bars are 10μm. Representative line scan traces across an APP + FE65 T579A cell and an APP + FE65 T579E cell are shown. ( E ) Subcellular fractionation of FE65 T579A, FE65 T579E, APP + FE65 T579A and APP + FE65 T579E from transfected CHO cells. The total lysates, cytosol/membrane fractions and nuclear fractions were probed with antibodies for APP and FE65. The purity of the fractions were revealed by probing with anti α-tubulin (cytosol/membrane) and c-jun (nuclei). The relative FE65 level in the cytosol/membrane fraction and nuclear fraction were analyzed by densitometry. n = 3. *P < 0.001. Results are means ± S.D.

Article Snippet: The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Immunofluorescence, Staining, Fractionation, Membrane