Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation by Casein Kinase 2 Facilitates Psh1 Protein-assisted Degradation of Cse4 Protein

doi: 10.1074/jbc.M114.580589

Figure Lengend Snippet: Deletion of CKA2 reduces cellular levels of polyubiquitylated Cse4. A , levels of Ub n -Cse4-Myc were measured in WT, psh1 Δ, and cka2 Δ strains with a deletion in PDR5. Cse4-Myc was expressed from its native promoter. An untagged strain was used as a control. Cultures were grown to midlog phase and treated with either MG132 (100 μ m ) or DMSO for 3.5 h. Anti-ubiquitin and anti-Myc Western blots of cell lysates confirmed the efficiency of proteasome inhibition by MG132 and similar Cse4-Myc levels, respectively ( left panel ). Polyubiquitylated proteins were pulled down from 2 mg of total proteins (from MG132-treated samples) using polyubiquitin affinity resin. Final elutions were probed with anti-ubiquitin and anti-Myc antibodies after SDS-PAGE ( right panel ). No obvious difference was detected in Ub n -Cse4-Myc levels among WT, psh1 Δ, and cka2 Δ strains. A nonspecific band is marked with an asterisk. B , Ub n -Cse4 levels are reduced in the cka2 Δ strain. WT, psh1 Δ, and cka2 Δ strains from A were used to perform a Cse4 ubiquitylation assay. EV indicates the empty vector control. After Gal induction of Cse4 for 2 h, cells were treated with either MG132 (100 μ m ) or DMSO for another 2 h. Anti-ubiquitin and anti-Cse4 Western blots of cell lysates confirmed the efficiency of proteasome inhibition by MG132 and similar Cse4 levels, respectively ( left panel ). Polyubiquitylated proteins were pulled down, and final elutions were analyzed as in A ( right panel ).

Article Snippet: Polyubiquitin affinity resin (Thermo, 89899) (30 μl of a 25% slurry) was added to 2 mg of total proteins diluted 1:1 with TBS and incubated for 3 h or overnight at 4 °C.

Techniques: Western Blot, Inhibition, SDS Page, Ubiquitin Assay, Plasmid Preparation

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: Isolation of endogenously polyubiquitinated protein complexes by use of the ubiquitin enrichment kit. A workflow for isolating endogenously ubiquitinylated protein complexes for proteomic analysis is shown. (A) Ubiquitinylated proteins were isolated from brain homogenates by IP. IP fraction (BF) and the supernatant (NBF) were loaded on an SDS-PAGE gel and stained for total protein expression (A1) and subsequently blotted on nitrocellulose membrane and stained with a polyclonal anti-Ub antibody (anti-Ub Ab) to determine enrichment efficiency (A2). (B) The IP fractions (BF and NBF) were further processed with the enrichment kit (Thermo) containing the polyubiquitin affinity resin (Poly-Ub AR). BF, NBF, and FT were loaded in SDS page (B1) and blotted with antiubiquitin antibody (B2). (C) Heat denaturation of high-affinity resin using high temperature (60°) was used to destroy the bond between resin and poly-Ub antibody. Poly-Ub was detected by performing a Western blot (agarose-only control; C1). BF, bound fraction; FT, flow-through; IP, immunoprecipitation; NBF, nonbound fraction; Poly-Ub, polymeric Ub; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Ub, ubiquitin.

Article Snippet: A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques: Isolation, SDS Page, Staining, Expressing, Western Blot, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: Poly-Ub levels. (A) Total levels of poly-Ub-bound proteins in DS (n = 6/group) and DS/AD (n = 6/group) cases compared with their age-matched controls (n = 6/group). (B, C) Increased poly-Ub of two types of ubiquitin chains [K63 (B) and K48 (C)] in both DS (n = 6/group) and DS/AD (n = 6/group) cases compared with their age-matched controls (n = 6/group). Representative bands are shown and protein levels (A−C, upper bands) were normalized per total protein load, named total load (A−C, lower bands). Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr y *p < 0.05; DS/AD vs. DS **p < 0.01; DS/AD vs. Ctr O ***p < 0.001; DS vs. Ctr Y and DS/AD vs. DS [for K48] *p < 0.05; DS vs. Ctr Y, DS/AD vs. Ctr O, and DS/AD vs. DS [for K63] **p < 0.01; Ctr O vs. Ctr Y #p < 0.05 one-way ANOVA). AD, Alzheimer's disease; ANOVA, analysis of variance; DS, Down syndrome; DS/AD, Down syndrome with Alzheimer's disease; SEM, standard error of the mean.

Article Snippet: A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: List of Identified Polyubiquitinated Proteins by Mass Spectrometry

Article Snippet: A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: MUbiSiDa Search

Article Snippet: A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: UCH-L1. (A, B) All samples (n = 6/group) were immunoprecipitated with anti-UCH-L1. Immunoprecipitated proteins were separated on SDS-PAGE, transferred on nitrocellulose membranes, and probed with anti-poly-UbK63 (A, upper bands) and anti-poly-UbK48 (B, upper bands). (A) Shows increased levels of poly-UbK63 bound to UCH-L1 in DS and DS/AD compared with their matched controls. (B) Shows an increase in poly-UbK48 bound to UCH-L1 in DS/AD compared with DS. All the IP experiments were normalized on the total amount of UCH-L1 (indicated as Expr.; A, B, lower bands). Representative bands are shown. Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr Y [for K63], DS/AD vs. DS [for K48] *p < 0.05; DS/AD vs. Ctr O [for K63] **p < 0.01 one-way ANOVA). UCH-L1, ubiquitin carboxyl-terminal hydrolase isozyme L1.

Article Snippet: A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques: Immunoprecipitation, SDS Page

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: Isolation of endogenously polyubiquitinated protein complexes by use of the ubiquitin enrichment kit. A workflow for isolating endogenously ubiquitinylated protein complexes for proteomic analysis is shown. (A) Ubiquitinylated proteins were isolated from brain homogenates by IP. IP fraction (BF) and the supernatant (NBF) were loaded on an SDS-PAGE gel and stained for total protein expression (A1) and subsequently blotted on nitrocellulose membrane and stained with a polyclonal anti-Ub antibody (anti-Ub Ab) to determine enrichment efficiency (A2). (B) The IP fractions (BF and NBF) were further processed with the enrichment kit (Thermo) containing the polyubiquitin affinity resin (Poly-Ub AR). BF, NBF, and FT were loaded in SDS page (B1) and blotted with antiubiquitin antibody (B2). (C) Heat denaturation of high-affinity resin using high temperature (60°) was used to destroy the bond between resin and poly-Ub antibody. Poly-Ub was detected by performing a Western blot (agarose-only control; C1). BF, bound fraction; FT, flow-through; IP, immunoprecipitation; NBF, nonbound fraction; Poly-Ub, polymeric Ub; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Ub, ubiquitin.

Article Snippet: Polyubiquitin affinity resin A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques: Isolation, SDS Page, Staining, Expressing, Western Blot, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: Poly-Ub levels. (A) Total levels of poly-Ub-bound proteins in DS (n = 6/group) and DS/AD (n = 6/group) cases compared with their age-matched controls (n = 6/group). (B, C) Increased poly-Ub of two types of ubiquitin chains [K63 (B) and K48 (C)] in both DS (n = 6/group) and DS/AD (n = 6/group) cases compared with their age-matched controls (n = 6/group). Representative bands are shown and protein levels (A−C, upper bands) were normalized per total protein load, named total load (A−C, lower bands). Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr y *p < 0.05; DS/AD vs. DS **p < 0.01; DS/AD vs. Ctr O ***p < 0.001; DS vs. Ctr Y and DS/AD vs. DS [for K48] *p < 0.05; DS vs. Ctr Y, DS/AD vs. Ctr O, and DS/AD vs. DS [for K63] **p < 0.01; Ctr O vs. Ctr Y #p < 0.05 one-way ANOVA). AD, Alzheimer's disease; ANOVA, analysis of variance; DS, Down syndrome; DS/AD, Down syndrome with Alzheimer's disease; SEM, standard error of the mean.

Article Snippet: Polyubiquitin affinity resin A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: List of Identified Polyubiquitinated Proteins by Mass Spectrometry

Article Snippet: Polyubiquitin affinity resin A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: MUbiSiDa Search

Article Snippet: Polyubiquitin affinity resin A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Polyubiquitinylation Profile in Down Syndrome Brain Before and After the Development of Alzheimer Neuropathology

doi: 10.1089/ars.2016.6686

Figure Lengend Snippet: UCH-L1. (A, B) All samples (n = 6/group) were immunoprecipitated with anti-UCH-L1. Immunoprecipitated proteins were separated on SDS-PAGE, transferred on nitrocellulose membranes, and probed with anti-poly-UbK63 (A, upper bands) and anti-poly-UbK48 (B, upper bands). (A) Shows increased levels of poly-UbK63 bound to UCH-L1 in DS and DS/AD compared with their matched controls. (B) Shows an increase in poly-UbK48 bound to UCH-L1 in DS/AD compared with DS. All the IP experiments were normalized on the total amount of UCH-L1 (indicated as Expr.; A, B, lower bands). Representative bands are shown. Densitometric values are shown as percentage of Ctr Y set as 100%. Mean ± SEM (DS vs. Ctr Y [for K63], DS/AD vs. DS [for K48] *p < 0.05; DS/AD vs. Ctr O [for K63] **p < 0.01 one-way ANOVA). UCH-L1, ubiquitin carboxyl-terminal hydrolase isozyme L1.

Article Snippet: Polyubiquitin affinity resin A Thermo Scientific Ubiquitin Enrichment Kit [89899] was used for the isolation and study of intracellular poly-Ub-modified proteins.

Techniques: Immunoprecipitation, SDS Page