Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Roles of Id1/HIF-1 and CDK5/HIF-1 in cell cycle reentry induced by amyloid-beta peptide in post-mitotic cortical neuron.

doi: 10.1016/j.bbamcr.2019.118628

Figure Lengend Snippet: Fig. 3. Aβ25-35 induces p25 production from cleavage of p35 without affecting CDK5 expression in cultured cortical neurons. Primary cortical cultures were treated with 10 μM Aβ25-35 for indicated times before detection of p35/p25 (A) or CDK5 (E) by Western blotting. The β-actin in (A) and α-tubulin in (E) served as the respective internal control for equal loading of proteins in each lane. Quantitative analyses of the signal intensities on the blots for p25, p35, p25/p35 ratio, and CDK5 are shown respectively in (B), (C), (D), and (F). Mean ± S.E.M. from N = 3 in all the statistical analyses. * denotes P < 0.05 compared with the corresponding control cultures without Aβ25-35 treatment in (B)–(D). The “ns” in (F) denotes “not significant”.

Article Snippet: The mouse antibody against β-actin (1:4000; Cat. No. MAB1501; Merck Millipore), mouse antibody against α-tubulin (1:10000; Cat. No. T9026; Sigma-Aldrich), rabbit antibody against cyclin D1 (1:1000; Cat. No. ab134175; Abcam), rabbit antibody against PCNA (1:500; Cat. No. ab92552; Abcam), rabbit antibody against HIF-1α (1:500; Cat. No. NB100-134; Novus Biologicals, Littleton, CO, USA), rabbit antibody against CDK5 (1:500; Cat. No. ab40773; Abcam), and rabbit antibody against p35/p25 (1:500; Cat. No. 2680; Cell Signaling Technology, Beverly, MA, USA) were all diluted in blocking buffer (5% nonfat dry milk in TBST buffer containing 0.05% Tween 20, 137 mM NaCl, and 20 mM Tris–HCl, pH 7.5).

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Roles of Id1/HIF-1 and CDK5/HIF-1 in cell cycle reentry induced by amyloid-beta peptide in post-mitotic cortical neuron.

doi: 10.1016/j.bbamcr.2019.118628

Figure Lengend Snippet: Fig. 5. Suppression of Id1 enhances p25 production, whereas exogenous Id1-Tag suppresses p25 production in rat cortical cultures. (A–F) Primary cortical cultures were transfected with sc siRNA or Id1 siRNA for 72 h followed by exposure to 10 μM Aβ25-35 for additional 4 h before detection of Id1 (A) or p35/p25 (C) by Western blotting. Quantitative analyses of the signal intensities on the blots for Id1, p25, p25/p35 ratio, and p35 are respectively shown in (B), (D), (E), and (F). Mean ± S.E.M. from N = 3 in all the statistical analyses. * and # denote P < 0.05. The “ns” denotes “not significant”. (G–L) Primary cortical cultures were treated with or without 4 μg/ml Id1-Tag for 2 h or 4 h before detection of p35/p25 (G) or CDK5 (K). Quantitative analyses of the signal intensities on the blots for p25, p25/ p35 ratio, p35, and CDK5 are shown respectively in (H), (I), (J), and (L). Mean ± S.E.M. from N = 4 in 2 h and N = 5 in 4 h. * denotes P < 0.05 as compared to the corresponding control cultures. The “ns” denotes “not significant”.

Article Snippet: The mouse antibody against β-actin (1:4000; Cat. No. MAB1501; Merck Millipore), mouse antibody against α-tubulin (1:10000; Cat. No. T9026; Sigma-Aldrich), rabbit antibody against cyclin D1 (1:1000; Cat. No. ab134175; Abcam), rabbit antibody against PCNA (1:500; Cat. No. ab92552; Abcam), rabbit antibody against HIF-1α (1:500; Cat. No. NB100-134; Novus Biologicals, Littleton, CO, USA), rabbit antibody against CDK5 (1:500; Cat. No. ab40773; Abcam), and rabbit antibody against p35/p25 (1:500; Cat. No. 2680; Cell Signaling Technology, Beverly, MA, USA) were all diluted in blocking buffer (5% nonfat dry milk in TBST buffer containing 0.05% Tween 20, 137 mM NaCl, and 20 mM Tris–HCl, pH 7.5).

Techniques: Transfection, Western Blot, Control

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Roles of Id1/HIF-1 and CDK5/HIF-1 in cell cycle reentry induced by amyloid-beta peptide in post-mitotic cortical neuron.

doi: 10.1016/j.bbamcr.2019.118628

Figure Lengend Snippet: Fig. 7. A diagram showing the proposed signal transduction pathway. We propose possible molecular mechanisms un- derlying Aβ25-35-dependent neuronal cell cycle reentry through Id1- and CDK5/p25-dependent HIF-1α induction in the fully differentiated post-mitotic cortical neurons. This mechanism is further complicated by the intriguing antag- onism between Id1 and p25 because they reciprocally sup- press expression of each other. Possible involvements of unknown mediators, as indicated by a red question mark, linking Aβ and HIF-1 is also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The mouse antibody against β-actin (1:4000; Cat. No. MAB1501; Merck Millipore), mouse antibody against α-tubulin (1:10000; Cat. No. T9026; Sigma-Aldrich), rabbit antibody against cyclin D1 (1:1000; Cat. No. ab134175; Abcam), rabbit antibody against PCNA (1:500; Cat. No. ab92552; Abcam), rabbit antibody against HIF-1α (1:500; Cat. No. NB100-134; Novus Biologicals, Littleton, CO, USA), rabbit antibody against CDK5 (1:500; Cat. No. ab40773; Abcam), and rabbit antibody against p35/p25 (1:500; Cat. No. 2680; Cell Signaling Technology, Beverly, MA, USA) were all diluted in blocking buffer (5% nonfat dry milk in TBST buffer containing 0.05% Tween 20, 137 mM NaCl, and 20 mM Tris–HCl, pH 7.5).

Techniques: Transduction, Expressing

Journal: BMC biology

Article Title: Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

doi: 10.1186/s12915-022-01312-4

Figure Lengend Snippet: Fig. 1 p35 interacts with JIP1. A Identification of JIP1 as p35-binding protein. Four peptides were identified and matched to mouse JIP1 by tandem mass spectrometry. B One microgram of bacterially expressed GST or GST fused-JIP1 was incubated with 2 μg His-p35 at 4°C for 2 h. The GST-tagged proteins were retrieved with GSH beads. The proteins were subjected to SDS-PAGE and detected using anti-p35 antibody by western blot. Amido black was used to visualize protein levels. C Endogenous p35 interacts with JIP1 in brain lysate. One microgram of IgG as control or JIP1 antibody was incubated with 500 μg of mouse brain lysate. Antibodies were pulled down using 50 μl goat-anti-rabbit IgG beads and the coupled proteins were subjected to SDS-PAGE followed by western blotting with anti-p35 antibody. D Confirmation of the specific interaction between p35 and JIP1 using p35 KO cortical neurons. Control IgG or anti-p35 was incubated with lysates from p35 WT or KO cortical neurons. The IPed proteins were immunoblotted with anti-JIP1 antibody. E Two-microgram control IgG or anti-p35 was incubated with lysate from JIP1 WT and KO neurons and the IPed proteins were immunoblotted with anti-JIP1 antibody. Images are representative of at least three independent experiments

Article Snippet: Mouse anti-JIP1 (B-7, Cat. #sc-25267, RRID: AB_627868), anti AIP4/Itch (G-11, Cat. #sc-28367, RRID: AB_667798), Rabbit anti-p35 (C64B10, 2680, C-19, Cat. #sc-820, RRID: AB_632137), Mouse anti-NUMB Antibody (48, Cat. #sc-136554, RRID: AB_10611794), Anti-c-Jun antibody (sc-74543, RRID: AB_1121646), and anti-Notch1 (mN1A, Cat. #sc-32745, RRID: AB_628033) were purchased from Santa Cruz Biotechnology (Texas, USA).

Techniques: Binding Assay, Mass Spectrometry, Incubation, SDS Page, Western Blot, Control

Journal: BMC biology

Article Title: Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

doi: 10.1186/s12915-022-01312-4

Figure Lengend Snippet: Fig. 2 JIP1 is a substrate of Cdk5. A Bacterially expressed GST-tagged JIP1 was subjected to in vitro kinase assay by Cdk5/p25 and 1 μCi of [γ-32P] ATP with or without Cdk5 inhibitor Roscovitine (Rosco) at 30°C for 30 min. The proteins were separated by SDS-PAGE for autoradiography. B, C Recombinant His-JIP1 was incubated with Cdk5/p25 with or without 20 μM of Roscovitine for the indicated duration. The proteins were subjected to western blot with anti-phospho-Serine (p-Ser) (B) or anti-phospho-Threonine (p-Thr). * and ** indicate full length of JIP1 and truncated form of JIP1 from C-terminus, respectively (C). Data representative of three independent experiments. D Schematic of domains of JIP1. Alignment of JIP1 protein sequence from different species analyzed by ClustaIW2 to show three conserved sites optimal for Cdk5 phosphorylation. E–G Identification of Cdk5 phosphorylation sites in JIP1. Ser197, Thr 205, and Ser 235 were all replaced with Ala. An in vitro kinase assay was carried out with purified active Cdk5/p35 incubated with WT-JIP1 (JIP1) or its Ala mutants, S197A (E), T205A (F), and S235A (G) and examined by western blot analysis using anti-phospho-Serine (p-Ser) or anti-phospho-Threonine (p-Thr). Data representative of three independent experiments. H Total cell lysate from p35 WT and KO neurons at DIV 3 was subjected to western blot analysis with custom pJIP1@T205 antibody (bands at ~100 and ~120 kDa, pJIP1@T205). Both bands from p35 WT or KO neurons were used for the measurement of JIP1 phosphorylation by Cdk5. I Quantification of phospho-JIP1 T205 levels relative to p35 WT neurons. WT, n=8 and KO, n=5, “n” equals the number of animals. Student’s t-test was used for statistical analysis and the data are presented as mean ± SEM. **** p < 0.0001

Article Snippet: Mouse anti-JIP1 (B-7, Cat. #sc-25267, RRID: AB_627868), anti AIP4/Itch (G-11, Cat. #sc-28367, RRID: AB_667798), Rabbit anti-p35 (C64B10, 2680, C-19, Cat. #sc-820, RRID: AB_632137), Mouse anti-NUMB Antibody (48, Cat. #sc-136554, RRID: AB_10611794), Anti-c-Jun antibody (sc-74543, RRID: AB_1121646), and anti-Notch1 (mN1A, Cat. #sc-32745, RRID: AB_628033) were purchased from Santa Cruz Biotechnology (Texas, USA).

Techniques: In Vitro, Kinase Assay, SDS Page, Autoradiography, Recombinant, Incubation, Western Blot, Sequencing, Phospho-proteomics, Purification

Journal: BMC biology

Article Title: Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

doi: 10.1186/s12915-022-01312-4

Figure Lengend Snippet: Fig. 5 JIP1 deficiency regulates axonal outgrowth via Notch1 intracellular domain. A Western blot analysis of Notch1 in total cell lysate from JIP1 WT and KO neurons at DIV 3. Graph represents quantification of Notch1 intracellular domain (~100kDa, Notch1-IC) levels in JIP1 WT and KO (n=6 each). Data presented as mean ± SEM and analyzed by Student’s t-test. “n” equals the number of animals. * p < 0.05. B Total cell lysate from p35 WT and KO neurons at DIV 3 subjected to western blot analysis with anti-Notch1 antibody. Quantification of Notch1 intracellular domain (Notch1-IC) levels in p35 WT (n=6) and KO (n=7) is presented as mean ± SEM and analyzed by Student’s t-test. “n” equals the number of animals. ** p < 0.01. C Representative images of JIP1 WT and KO neurons infected with adenovirus carrying GFP or Numb. D Data was analyzed by two-way ANOVA and presented as mean ± SEM; n=163 for GFP, n=132 for Numb for JIP1 WT neurons and n=248 for GFP, n=247 for Numb for JIP1 KO neurons. “n” equals the number of neurons from one experiment. **** p < 0.0001. E JIP1 WT and KO neurons immunoprecipitated with Notch1 antibody followed by immunoblotting with ubiquitin (Ub) antibody

Article Snippet: Mouse anti-JIP1 (B-7, Cat. #sc-25267, RRID: AB_627868), anti AIP4/Itch (G-11, Cat. #sc-28367, RRID: AB_667798), Rabbit anti-p35 (C64B10, 2680, C-19, Cat. #sc-820, RRID: AB_632137), Mouse anti-NUMB Antibody (48, Cat. #sc-136554, RRID: AB_10611794), Anti-c-Jun antibody (sc-74543, RRID: AB_1121646), and anti-Notch1 (mN1A, Cat. #sc-32745, RRID: AB_628033) were purchased from Santa Cruz Biotechnology (Texas, USA).

Techniques: Western Blot, Infection, Immunoprecipitation, Ubiquitin Proteomics

Journal: BMC biology

Article Title: Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

doi: 10.1186/s12915-022-01312-4

Figure Lengend Snippet: Fig. 6 Phosphorylated JIP1 enhances Itch phosphorylation by Cdk5 to promote Notch1-IC ubiquitination. A Western blot analysis of phospho-Itch (p-Itch@T222) and Itch (Itch) in total cell lysate from JIP1 WT and KO neurons (DIV 3). Graph represents quantification of phospho-Itch levels relative to Itch in JIP1 WT and KO (n=4 each). Groups were compared using Student’s t-test and data presented as mean ± SEM. “n” equals the number of animals. ** p < 0.01. B Western blot analysis of in vitro binding assay using bacterially expressed GST or GST fused-p35 incubated with His-JIP1 and/or Itch recombinant proteins at 4 °C for 2 h. GST-tagged proteins were retrieved with GSH beads. The proteins were immunoblotted with Itch and JIP1 antibody. Data representative of three independent experiments. C Immunoblot analysis of CD-1 embryonic cortex lysate (E15.5-16.5) immunoprecipitated with Itch antibody and probed with JIP1 antibody. Data representative of three independent experiments. D Western blot analysis of Itch phosphorylation by Cdk5. Recombinant Itch protein incubated with Cdk5/p35 with or without 20 μM of Roscovitine (Rosco) at 30 °C for 2 h was immunoblotted with phospho-Itch (pItch@T222) and Itch antibody. Data representative of three independent experiments. E Western blot analysis of the contribution of JIP phosphorylation to Itch phosphorylation. Recombinant JIP1 or T205A was incubated with Cdk5/p35 at 30 °C for 1 h followed by the addition of Itch and incubated for 15 min. The mixture was then analyzed by immunoblotting with phospho-Itch and Itch antibody. F Quantification of phospho-Itch levels in E. Multiple comparison was analyzed by one-way ANOVA and data are presented as mean ± SEM from four independent experiments. * p < 0.05. G Wild-type Itch (WT) or Itch mutants (phosphomimic T222D (T222D) or Ala mutant T222A (T222A)) were co-transfected into HEK293A cells with Notch1-IC-Flag and immunoprecipitated with Notch1 antibody. Immunoprecipitated proteins were subjected to western blot analysis with Ubiquitin (UB) antibody. Data representative of three independent experiments

Article Snippet: Mouse anti-JIP1 (B-7, Cat. #sc-25267, RRID: AB_627868), anti AIP4/Itch (G-11, Cat. #sc-28367, RRID: AB_667798), Rabbit anti-p35 (C64B10, 2680, C-19, Cat. #sc-820, RRID: AB_632137), Mouse anti-NUMB Antibody (48, Cat. #sc-136554, RRID: AB_10611794), Anti-c-Jun antibody (sc-74543, RRID: AB_1121646), and anti-Notch1 (mN1A, Cat. #sc-32745, RRID: AB_628033) were purchased from Santa Cruz Biotechnology (Texas, USA).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Western Blot, In Vitro, Binding Assay, Incubation, Recombinant, Immunoprecipitation, Comparison, Mutagenesis, Transfection

Journal: BMC biology

Article Title: Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

doi: 10.1186/s12915-022-01312-4

Figure Lengend Snippet: Fig. 7 Illustration of Cdk5-JIP1-Notch1 axis in regulation of axonal outgrowth. A Cdk5/p35-mediated JIP1 phosphorylation boosts phosphorylation of Itch by Cdk5, which facilitates proteasome degradation of Notch1, resulting in enhanced axonal outgrowth. B Loss of JIP1 or p35 leads to an accumulation of Notch1, which result in inhibition of axonal outgrowth

Article Snippet: Mouse anti-JIP1 (B-7, Cat. #sc-25267, RRID: AB_627868), anti AIP4/Itch (G-11, Cat. #sc-28367, RRID: AB_667798), Rabbit anti-p35 (C64B10, 2680, C-19, Cat. #sc-820, RRID: AB_632137), Mouse anti-NUMB Antibody (48, Cat. #sc-136554, RRID: AB_10611794), Anti-c-Jun antibody (sc-74543, RRID: AB_1121646), and anti-Notch1 (mN1A, Cat. #sc-32745, RRID: AB_628033) were purchased from Santa Cruz Biotechnology (Texas, USA).

Techniques: Phospho-proteomics, Inhibition

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 1. The expression of Vps35, p35, CDK5, and tau s396 in retina and primary cultured RGCs. (A, B) Vps35 and p35 were colo- calized in the GCL and the signals of Vps35 decreased while the signals of p35 increased 1 week after intravitreal injection of 50 nmol glutamate. (C, D) Vps35 and p35, and Vps35 and tau s396 colocalized in the GCL. (E, F) The expression of p35 and Cdk5 in primary cultured RGCs. (G, H) The colocalization of Vps35 and p35, Vps35 and tau s396, p35 and Cdk5, and Vps35 and Cdk5 in primary cultured RGCs.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Expressing, Cell Culture, Injection

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 2. The relative mRNA and protein expression of Vps35 decreased, while the expression of p35, p-tau s396 increased with glutamate excitotoxity both in vivo (especially 7D and 14D after intravitreal injection of glutamate 50 nmol) (A–C) and in vitro (D, E). **P < 0.01; *P < 0.05.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Expressing, In Vivo, Injection, In Vitro

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 3. Vps35 alters the activity of Cdk5/p35 and the phosphorylation of tau protein. (A, B, E) Downregulation of Vps35 in primary cultured RGC could statistically enhance the levels of Cdk5/p35 and tau s396. (C, D, F) Overexpression of Vps35 in primary cultured RGC could considerably decrease the levels of Cdk5/p35 and tau s396. (G, H) Roscovitine significantly inhibited Cdk5 (P < 0.05), resulting in a decrease in the p35 and p-tau s396 levels in primary cultured RGCs. **P < 0.01; *P < 0.05. DMSO, dimethylsulfoxide.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Activity Assay, Phospho-proteomics, Cell Culture, Over Expression

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 5. Vp35 interacts with p35 in vivo and in vitro. (A, B) Vps35 and p35 were colocalized in the cytoplasm of RGCs in vivo and in vitro by immunofluorescence. (C) The colocalization of Cdk5 and p35 in primary cultured RGCs. (D) The interaction between Vps35 and p35 by IP was obvious 1 week after the intravitreal injection of 50 nmol glutamate. Cdk5 interacted with p35 both in control group and one week after the intravitreal injection of 50 nmol of glutamate.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: In Vivo, In Vitro, Immunofluorescence, Cell Culture, Injection, Control

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 6. Deficiency of Vps35 leads to a decrease in LAMP1 of retina. (A, B) The expression of LAMP1 and EEA1 in the retina significantly decreased, while p35 increased, 1 week after the intravitreal injection of 50 nmol of glutamate. (C, D) The inhibition of Vps35 resulted in a decrease in LAMP1 in primary RGCs. **P < 0.01; *P < 0.05.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Expressing, Injection, Inhibition

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 7. There was a direct interaction between Vps35 and LAMP1 and between LAMP1 and p35. (A–C) The colocalization of Vps35 and LAMP1, p35 and LAMP1, and p35 and EEA1 in the GCL of retina. (D–F) The colocalization of Vps35 and LAMP1, p35 and LAMP1, and p35 and EEA1 in primary cultured RGC. (G) The coimmunoprecipitation showed a direct interaction of Vps35 and LAMP1, and LAMP1 and p35, with protein extracted from rat retinae.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Cell Culture