Journal: F1000Research

Article Title: The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.132249.1

Figure Lengend Snippet: Summary of the Profilin-1 antibodies tested.

Article Snippet: Aviva Systems Biology , ARP48269 , QC20112-42607 , AB_1294625 , polyclonal , - , rabbit , 0.5 , Wb.

Techniques: Concentration Assay, Recombinant

Journal: Aging Cell

Article Title: Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

doi: 10.1111/acel.13573

Figure Lengend Snippet: APP‐KCC2 interaction is enhanced by gamma frequency light flicker to stabilize KCC2 on the plasma membrane. (a) Representative immunoblots of surface KCC2 and GABA A R α1 levels in 6‐month‐old WT or APP/PS1 mice under 7 days of 1 h/day 40 Hz light flicker or not ( n = 3 mice per group). Data are presented as mean ± SEM. # p < 0.05 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (b) Quantification of surface‐KCC2 levels. (c) Quantification of surface‐GABA A R α1 levels. (d) Representative Western blots showing co‐immunoprecipitation with both KCC2 and APP antibodies in cerebral cortex of 6‐month‐old WT or APP/PS1 mice with or without 40 Hz light flicker ( n = 3 mice per group). Data are presented as mean ± SEM. # p < 0.05 vs. indicated group, ## p < 0.01 vs. indicated group, by unpaired t ‐test. (e) Relative immunoreactivity of APP normalized to KCC2 (IP: KCC2). (f) Relative immunoreactivity of KCC2 normalized to APP (IP: APP). (g) Immunohistochemistry with anti‐APP (red) and KCC2 (green) in cerebral cortex of 6‐month‐old WT or APP/PS1 under 7 days of 1 h/day 40 Hz light flicker or not. Scale bar, 50 μm. (h) Pearson's correlation coefficient analysis of APP and KCC2, and quantification of KCC2 levels in different groups ( n = 18 slices from 7 to 9 mice per group). Data are presented as mean ± SEM. *p < 0.05 vs. WT group, **p < 0.01 vs. WT group; #p < 0.05 vs. indicated group, ##p < 0.01 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (i) Representative immunoblots of surface KCC2, GABA A R α1, and APP levels in siNC, siKCC2, and siAPP treatment group. (j) Quantification of surface‐KCC2, surface‐GABA A R α1, surface‐APP levels ( n = 3). Data are presented as mean ± SEM. *p < 0.05 vs. control group, **p < 0.01 vs. control group; #p < 0.05 vs. indicated group, ##p < 0.01 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test

Article Snippet: KCC2 siRNA , Santa Cruz Biotechnology , Cat# sc‐42607.

Techniques: Western Blot, Immunoprecipitation, Immunohistochemistry

Journal: Aging Cell

Article Title: Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

doi: 10.1111/acel.13573

Figure Lengend Snippet: Gamma frequency light flicker suppresses KCC2 internalization and subsequent degradation via regulating both tyrosine phosphorylation and ubiquitination, leading to an increase in surface‐KCC2 levels. (a) Cortex extracted from 6‐month‐old WT and APP/PS1 littermates treated with 7 days of 1 h/day dark or 40 Hz light flicker immunoprecipitated with an anti‐KCC2 antibody (IP: KCC2) and probed with anti‐ubiquitin antibody. (b) Quantification of the ubiquitinated KCC2 (Ub‐KCC2) for each group ( n = 3 mice per group). Data are presented as mean ± SEM. *p < 0.05 vs. WT group, #p < 0.05 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (c) Cortex isolated from 6‐month‐old WT and APP/PS1 littermates with or without 7 days of 1 h/day 40 Hz light flicker immunoprecipitated with an anti‐KCC2 antibody (IP: KCC2) and probed with anti‐phospho‐Tyrosine antibody. (d) Quantification of phosphorylated KCC2 on tyrosine (p‐KCC2 (Tyr)) for each group ( n = 3 mice per group). Data are presented as mean ± SEM. #p < 0.05 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (e) Representative immunoblots of KCC2 incubated with MG132 at different concentrations. (f) Representative immunoblots of membrane proteins from 6‐month‐old WT or APP/PS1 mice treated with or without 7 days of 1 h/day 40 Hz light flicker and MG132. (g) Relative immunoreactivity of surface‐KCC2 normalized to Na/K‐ATPase ( n = 3). Data are presented as mean ± SEM. *p < 0.05 vs. control group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test

Article Snippet: KCC2 siRNA , Santa Cruz Biotechnology , Cat# sc‐42607.

Techniques: Immunoprecipitation, Isolation, Western Blot, Incubation

Journal: Aging Cell

Article Title: Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

doi: 10.1111/acel.13573

Figure Lengend Snippet: Activated PKC by gamma frequency light flicker phosphorylates APP and KCC2 to maintain membrane levels of both, which contributes to the upregulation of surface‐GABA A R α1. (a) Representative immunoblots showing levels of p‐PKC in cortex of 6‐month‐old APP/PS1 mice after 7 days of 1 h/day dark, 40 Hz flicker, RO 31‐8220 (6 mg/kg/d, s.c), RO 31‐8220 (6 mg/kg/d, s.c) with 40 Hz flicker. Immunoprecipitates were analyzed to detect the serine phosphorylation levels of APP and KCC2 with anti‐KCC2, anti‐APP, and anti‐phosphoserine antibodies. (b) Quantification of phosphorylated KCC2 and APP normalized to total KCC2 and APP ( n = 4 mice per group). Data are presented as mean ± SEM. *p < 0.05 vs. APP/PS1 group; #p < 0.05 vs. indicated group; by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (c) Soluble and insoluble Aβ 1‐40 and Aβ 1‐42 levels in cortex of APP/PS1 mice exposed to dark, 40 Hz flicker, RO 31‐8220 (6 mg/kg/d, s.c), RO 31‐8220 (6 mg/kg/d, s.c) with 40 Hz flicker were performed by ELISA (8 mice/group). Data are presented as mean ± SEM. **p < 0.01 vs. APP/PS1 group; ##p < 0.01 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (d) Representative immunoblots showing co‐immunoprecipitation with both KCC2 and APP antibodies in cortex of APP/PS1 mice exposed to dark, 40 Hz flicker, RO 31‐8220 (6 mg/kg/d, s.c), RO 31‐8220 (6 mg/kg/d, s.c) with 40 Hz flicker ( n = 6 mice/group). Data are presented as mean ± SEM. *p < 0.05 vs. APP/PS1 group; **p < 0.01 vs. APP/PS1 group; #p < 0.05 vs. indicated group; ##p < 0.01 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (e) Relative immunoreactivity of APP normalized to KCC2 (IP: KCC2). (f) Relative immunoreactivity of KCC2 normalized to APP (IP: APP). (g) Representative immunoblots of membrane proteins from 6‐month‐old APP/PS1 mice exposed to 7 days of dark, 40 Hz flicker, RO 31‐8220 (6 mg/kg/d, s.c), RO 31‐8220 (6 mg/kg/d, s.c) with 40 Hz flicker (3 mice per group). Data are presented as mean ± SEM. *p < 0.05 vs. APP/PS1 group; ##p < 0.01 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (h) Immunohistochemistry with anti‐APP (red) and anti‐KCC2 (green) in cortex of 6‐month‐old APP/PS1 treated with dark, 40 Hz light flicker, RO 31‐8220 (6 mg/kg/d, s.c), RO 31‐8220 (6 mg/kg/d, s.c) with 40 Hz flicker for 7 days ( n = 5 mice/group). Scale bar, 50 μm. (i) Gates P2 (green gate) and P3 (orange gate) for surface APP and GABA A R α1 were determined, respectively, in the unstained group, and the number of APP + cells (gate P2) was allowed to count 10,000 statistically in each experimental group, and the percentage number of GABA A R α1 + cells and mean fluorescence intensity (MFI) levels of surface GABA A R α1 in the gate P2 (APP + cells) were analyzed on a CytoFLEX flow cytometer, using CytExpert software ( n = 5 mice/group). Data are presented as mean ± SEM. *p < 0.05 vs. APP/PS1 group; **p < 0.01 vs. APP/PS1 group; #p < 0.05 vs. indicated group, by two‐way ANOVA with Tukey's post hoc multiple comparisons test. (j) Immunohistochemistry with anti‐Aβ (green) and anti‐EEA1 (red) antibodies in cortex of 6‐month‐old APP/PS1 treated with dark, 40 Hz light flicker, RO 31‐8220 (6 mg/kg/d, s.c), RO 31‐8220 (6 mg/kg/d, s.c) with 40 Hz flicker for 7 days ( n = 6 to 7 mice per group), scale bar, 50 μm. DAPI labeling was used for cell nuclei

Article Snippet: KCC2 siRNA , Santa Cruz Biotechnology , Cat# sc‐42607.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Immunohistochemistry, Fluorescence, Flow Cytometry, Software, Labeling

Journal: Aging Cell

Article Title: Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

doi: 10.1111/acel.13573

Figure Lengend Snippet: Model shows the potential mechanism by which 40 Hz light flicker reduces Aβ levels. Phosphorylation of APP induced by PKC activation under the treatment of 40 Hz light flicker led to maintained plasma membrane levels of full‐length APP as well as decreased trafficking to endosomes, which ultimately inhibited BACE1 cleavage pathway. Moreover, on the basis of PKC‐induced serine phosphorylation of KCC2, the tyrosine phosphorylation and degradation of KCC2 were further limited by a direct interaction with full‐length APP anchored within the plasma membrane, which contributed to the upregulation of surface GABA A receptor α1 levels. In addition, the increase of ATP caused by 40 Hz light flicker promoted PLC/DAG signaling cascade, which is likely to be involved in the activation of PKC

Article Snippet: KCC2 siRNA , Santa Cruz Biotechnology , Cat# sc‐42607.

Techniques: Activation Assay

Journal: Aging Cell

Article Title: Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

doi: 10.1111/acel.13573

Figure Lengend Snippet: List of reagent or resource used in this study

Article Snippet: KCC2 siRNA , Santa Cruz Biotechnology , Cat# sc‐42607.

Techniques: Plasmid Preparation, ATP Assay, Enzyme-linked Immunosorbent Assay, Isolation, Cell Fractionation