usp7 polyclonal antibody  (Proteintech)

Journal: eLife

Article Title: The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5

doi: 10.7554/eLife.79013

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-USP7 (Rabbit polyclonal) , Thermo Fisher Scientific , Cat# A300-033A, RRID: AB_203276 , WB (1:1000).

Techniques: Recombinant, Expressing, Purification, Bacteria, Ubiquitin Proteomics, Methylation

Journal: eLife

Article Title: The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5

doi: 10.7554/elife.79013

Figure Lengend Snippet: Figure 2. PAF15 associates with the TRAF and UBL1-2 domains of USP7. (A) Schematic illustration of PAF15-USP7 binding experiment. USP7 recognizes P/A/ExxS or KxxxK motifs in its substrates via TRAF or ubiquitin-like (UBL) domains, respectively. PAF15 has three motifs, and alanine mutations were introduced at S79, S97, K101, and K105. (B) GST pull-down from the interphase egg extracts using GST-PAF15, P/AxxS mutant (S79A/S97A; SA), KxxxK mutant (K101A/K105A; KA), and triple mutant (SAKA). The samples were analyzed by immunoblotting using the antibodies indicated. Purified GST or GST-PAF15 mutants used in pull-down assay were stained using CBB. (C) Schematic illustration of rUSP7 truncation mutants employed in (D). (D) FLAG pull-down from interphase egg extracts using rUSP7-3xFLAG mutants presented in (C), W167A and 3 A point mutants. USP7 3 A: D780A/E781A/ D786A. The samples were analyzed by immunoblotting using the antibodies indicated. Samples were also stained by CBB. Arrowheads indicate rUSP7 truncation mutants and point mutants. Source data are provided as Figure 2—source data 1.

Article Snippet: Rabbit polyclonal USP7 antibody (A300- 033A) was purchased from Bethyl Laboratories.

Techniques: Binding Assay, Ubiquitin Proteomics, Mutagenesis, Western Blot, Purification, Pull Down Assay, Staining

Journal: eLife

Article Title: The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5

doi: 10.7554/elife.79013

Figure Lengend Snippet: Figure 7. PAF15 dissociation negatively regulates aberrant increase of DNA methylation. Sperm chromatin and radiolabeled S-(methyl-3H)-adenosyl-L- methionine were added to either Mock- and USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts. Purified DNA samples were analyzed to determine the efficiency of DNA methylation. Data are presented as mean ± SEM from three biological replicates. Multiple comparisons were performed by two- way repeated measure ANOVA (RM ANOVA) followed by Sidak’s multiple comparison test. ns; not significant, ∗p<0.05, ∗∗∗p<0.001, and ∗∗∗∗p<0.0001. Source data are provided as Figure 7—source data 1.

Article Snippet: Rabbit polyclonal USP7 antibody (A300- 033A) was purchased from Bethyl Laboratories.

Techniques: DNA Methylation Assay, Purification, Comparison

Journal: eLife

Article Title: The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5

doi: 10.7554/elife.79013

Figure Lengend Snippet: Figure 8. mUSP7 interacts with mPAF15 to assure a complete DNA methylation maintenance. (A) Immunoprecipitation (IP) of endogenous mPAF15 from whole cell lysates of wt and mPAF15 SAKA mouse embryonic stem cells (mESCs) using an anti-PAF15 antibody. Bound fractions were subjected to immunoblotting with anti-USP7 and PAF15 antibodies. The bar plot shows the quantifications of the relative GFP-mUSP7 and mUSP7 co-precipitated with mPAF15. The error bar stands for SD from three biological replicates. A paired t-test with two tails was done, and p value was indicated. (B) Scheme of the cell fractionation experiment described in Figure 7B (left). IP of endogenous mPAF15 from cytosolic, soluble nuclear, and chromatin fractions using an anti-PAF15 antibody. Bound fractions were subjected to immunoblotting with PAF15 antibody. Immunoblotting with anti-Tubulin and anti-H3 antibodies is used to indicate the cytosolic and chromatin fractions, respectively. (C) Boxplot shows the DNA methylation levels of LINE-1 elements in both wt and mPAF15 SAKA naïve and epiblast-like cells (EpiLCs). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Data sets from four biological replicates were tested for significance with an unpaired t-test with one tail was performed, and p values are indicated.

Article Snippet: Rabbit polyclonal USP7 antibody (A300- 033A) was purchased from Bethyl Laboratories.

Techniques: DNA Methylation Assay, Immunoprecipitation, Western Blot, Cell Fractionation, Software