anti ube2e3  (Proteintech)

Journal: PeerJ

Article Title: UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging

doi: 10.7717/peerj.12253

Figure Lengend Snippet: (A) The expression of UBE2E3 in 7862 normal tissues of GTEx. (B) The correlation analysis of UBE2E3 with osteogenesis related genes in GTEx. (C) The correlation analysis of COL1A1 and UBE2E3 in GTEx. (D, E) Heatmap of single cell sequencing of rat BMSCs. (F) UBE2E3 expression of human BMSCs from GSE35956 and GSE35958 . (G) qRT-PCR analyses of UBE2E3 levels in old mouse BMSCs and young mouse BMSCs. (H) Western blot analyses of UBE2E3 levels in old mouse BMSCs and young mouse BMSCs. (I) qRT-PCR analyses of UBE2E3 levels in increasing numbers of passages of mice BMSCs. (J) Western blot analyses of UBE2E3 levels in increasing numbers of passages of mice BMSCs. Error bars showed standard deviation. * P < 0.05, *** P < 0.001.

Article Snippet: Next, membranes were blocked at room temperature with 5% defatted milk for 1 h. Then the membrane was incubated with anti- UBE2E3 (bs-8352R, 1:500; Bioss), anti- GAPDH (10494-1-AP,1:5000; Proteintech) overnight at 4 °C.

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: PeerJ

Article Title: UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging

doi: 10.7717/peerj.12253

Figure Lengend Snippet: (A) Knockdown efficiency of UBE2E3 was verified by qRT-PCR. (B) Knockdown efficiency of UBE2E3 was verified by western blot. (C) β-Gal staining and quantification of senescent BMSCs in siUBE2E3 group and siNC group. (D) The relative mRNA expression of p16 and p21 between siUBE2E3 group and siNC group. (E) ALP and ARS staining results in siUBE2E3 group and siNC group, with the quantitative analysis. (F) The relative mRNA expression of RUN2, ALP, SP7 and BGLAP between siUBE2E3 group and siNC group. Error bars showed standard deviation. * P < 0.05, ** P < 0.01.

Article Snippet: Next, membranes were blocked at room temperature with 5% defatted milk for 1 h. Then the membrane was incubated with anti- UBE2E3 (bs-8352R, 1:500; Bioss), anti- GAPDH (10494-1-AP,1:5000; Proteintech) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Expressing, Standard Deviation

Journal: PeerJ

Article Title: UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging

doi: 10.7717/peerj.12253

Figure Lengend Snippet: (A) Overexpression efficiency of UBE2E3 was verified by qRT-PCR. (B) Overexpression efficiency of UBE2E3 was verified by western blot. (C) β-Gal staining and quantification of senescent BMSCs in UBE2E3 overexpression group and the control group. (D) The relative mRNA expression of p16 and p21 between UBE2E3 overexpression group and the control group. (E) ALP and ARS staining results in UBE2E3 overexpression group and the control group, with the quantitative analysis. (F) The relative mRNA expression of RUN2, ALP, SP7 and BGLAP between UBE2E3 overexpression group and the control group. Error bars showed standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Next, membranes were blocked at room temperature with 5% defatted milk for 1 h. Then the membrane was incubated with anti- UBE2E3 (bs-8352R, 1:500; Bioss), anti- GAPDH (10494-1-AP,1:5000; Proteintech) overnight at 4 °C.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Staining, Expressing, Standard Deviation

Journal: PeerJ

Article Title: UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging

doi: 10.7717/peerj.12253

Figure Lengend Snippet: (A) The correlation of NQO1 and UBE2E3 in GSE35956 . (B) The correlation analysis of GCLC and UBE2E3 in GSE35956 . (C) The correlation analysis of GCLC and UBE2E3 in GSE35958 . (D) The correlation of GCLM and UBE2E3 in GSE35958 . (E) Representative images of BMSCs treated with siNC and siUBE2E3, and stained with DAPI and antibody against Nrf2. Scale bar, 50 µm. (F) NQO1, GCLC and GCLM expression between siUBE2E3 group and siNC group as analyzed by qRT-PCR. Error bars showed standard deviation. * P < 0.05, *** P < 0.001.

Article Snippet: Next, membranes were blocked at room temperature with 5% defatted milk for 1 h. Then the membrane was incubated with anti- UBE2E3 (bs-8352R, 1:500; Bioss), anti- GAPDH (10494-1-AP,1:5000; Proteintech) overnight at 4 °C.

Techniques: Staining, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: PeerJ

Article Title: UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging

doi: 10.7717/peerj.12253

Figure Lengend Snippet: High expression of UBE2E3 contributes to nuclear translation of Nrf2 and its high activity. With age, the expression of UBE2E3 decreases and nuclear accumulation of Nrf2 and its activity are restrained, leading to cell senescence and reduced osteogenic differentiation potential of BMSCs.

Article Snippet: Next, membranes were blocked at room temperature with 5% defatted milk for 1 h. Then the membrane was incubated with anti- UBE2E3 (bs-8352R, 1:500; Bioss), anti- GAPDH (10494-1-AP,1:5000; Proteintech) overnight at 4 °C.

Techniques: Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Genes regulating membrane-associated E-cadherin and proliferation in adenomatous polyposis coli mutant colon cancer cells: High content siRNA screen

doi: 10.1371/journal.pone.0240746

Figure Lengend Snippet: SW480 cells were transfected with individual siRNA duplexes from the UBE2E3 SMARTpool for 72 hours: (A) Expression of ZEB1, E-cadherin and UBE2E3 were analysed by immunoblot. β-tubulin was used as a loading control. siRNA#1 (1*) shares the same 5’ nucleotide sequence as miR200 family seed sequence. The blot is representative of three individual experiments. Shown are cropped images, uncropped blots are included in ; (B) Quantitation of E-cadherin, ZEB1 and UBE2E3 protein levels upon siUBE2E3 knockdown in SW480 cells. Protein levels were determined using densitometry against the loading control β-tubulin and are representative for triplicate experiments (mean± SEM) *p<0.05 (p = 0.026 and p = 0.039 for si UBE2E3 1 and 3, respectively), **p<0.005 (p = 0.00247), ***p<0.001 for E-cadherin and UBE2E3 or duplicate experiments (mean ± sd) for ZEB1 *p = 0.023, **p = 0.0035, ***p<0.001; one-tailed unpaired t -test vs mock control; (C) Immunofluorescence staining of E-cadherin in fixed SW480 cells, 72 hours post treatment with si UBE2E3 siRNA duplexes #1, 2, 3 or mock control. Scale bar; 50μM.

Article Snippet: The following primary and secondary antibodies were used: anti-E-cadherin (Abcam HECD1 #ab1416 Lot# GR91484-1, 1:200), anti-E-cadherin (Cell Signalling 24E10 #3195 Lot# 04/2014, 1:1000), anti-E-cadherin (BD Transduction Laboratories #610182 Lot# 316522, 1:1000), anti-ZEB1 (Santa Cruz H-102 #sc-25388, Lot# H1513 1:1000), anti-UBE2E3 (Abcam 4B4 #ab128098 (OT14B4) Lot# GR45436-1, 1:1000), anti-SNX27 (Abcam 1C6 #ab77799 Lot# GR212908-1 and GR20549-1, 1:500), anti-MMP14 (Millipore #MAB3328 LEM-2/15.8 Lot# 2488951, 1:1000), anti-ZO1 (BD Transduction Laboratories #61096, Lot# 34962 1:200), anti-β-tubulin (Sigma Aldrich TUB2.1 T4026 Lot# 125M4884V, 1:1000), anti-occludin (Abcam #ab31721 Lot# GR115633-1, 1:200), Alexa488 goat αmouse/rabbit (#A-11001 and #A-11035 Thermo Scientific, 1:500) and Alexa546 goat αmouse/rabbit (#A-11030 and #A-11035 Thermo Scientific, 1:500).

Techniques: Transfection, Expressing, Western Blot, Sequencing, Quantitation Assay, One-tailed Test, Immunofluorescence, Staining

Journal: Neuropathology

Article Title: Praja1 RING ‐finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP ‐43 aggregate formation

doi: 10.1111/neup.12694

Figure Lengend Snippet: PCR primers used in this study

Article Snippet: The blotted membrane was blocked with 3% skim milk and incubated overnight with rabbit anti‐FLAG (#7425; Sigma), rabbit anti‐TDP‐43 C‐terminus (405‐414) (#TIP‐TDP09; Cosmo Bio, Tokyo, Japan), rabbit anti‐phosphorylated TDP‐43 (pSer409/S410) (#TIP‐PTD‐P02; Cosmo Bio), rabbit anti‐PJA1 (#17687‐1‐AP; ProteinTech Japan, Tokyo, Japan), rabbit anti‐Myc Tag (#2278; Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐ubiquitin K48 (#05‐1307; Sigma), rabbit anti‐UBE2D2/3 (#HPA003921: Sigma), rabbit anti‐UBE2E3 (#15488‐1‐AP; ProteinTech), rabbit anti‐UBE2K (#11834‐3‐AP; ProteinTech), and mouse monoclonal anti‐GAPDH (#ab8245; Abcam, Cambridge, UK) antibodies at dilutions of 1:1000, followed by incubation with biotinylated anti‐rabbit or anti‐mouse IgG (Vector Laboratories, Burlingame, CA, USA; 1:1000) for 1 h and streptavidin‐alkaline phosphatase (#11089161001; Sigma; 1:1000) for 1 h. Reactions were visualized by color development using nitroblue tetrazolium chloride (NBT) and 5‐bromo‐4‐chloro‐3‐indolylphosphate p‐toluidine salt (BCIP) (#11681451001; Sigma).

Techniques:

Journal: Neuropathology

Article Title: Praja1 RING ‐finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP ‐43 aggregate formation

doi: 10.1111/neup.12694

Figure Lengend Snippet: PJA1 binding to TDP‐43 and E2 ubiquitin conjugating enzyme UBE2E3. (A) Western blot analysis of suppressive effects of adenovirus expressing human PJA1v1, but not PJA1ΔR, on phosphorylation and aggregate formation of TDP‐43 after DsRed‐ and FLAG‐tagged wild‐type (WT) and CTF TDP‐43 adenovirus infection in the presence of 0.5 μM MG‐132 (lanes 3–8). The CTF TDP‐43 adenovirus preferably induces RIPA‐insoluble phosphorylated TDP‐43 (lanes 4, 6). Adenovirally‐induced hPJA1v1 and hPJA1ΔR is consistently localized in both RIPA‐soluble and insoluble fractions (lanes 3, 5, 7, 8), and adenoviral hPJA1v1, but not hPJA1ΔR, decreases RIPA‐insoluble phosphorylated CTF TDP‐43 (lanes 5, 7) in a similar manner to the experiment shown in Figure . Endogenous UBE2E3 is also detected both in RIPA‐soluble and insoluble fractions. (B) The co‐immunoprecipitation (Co‐IP) assay shows that PJA1 preferentially binds to CTF TDP‐43 rather than WT TDP‐43 (PJA1 blot; lanes 3, 5, 7, 8), while CTF TDP‐43 is consistently ubiquitinated irrespective of adenoviral PJA1 induction (Ubiquitin K48 blot; lanes 4–8). Both native and adenoviral UBE2E3 are co‐immunoprecipitated with WT and CTF TDP‐43 (UBE2E3 and Myc‐Tag blots). (C) Co‐IP assay indicates that TDP‐43 and/or PJA1 bind to UBE2E3 but not UBE2D2/3 or UBE2K. (D) Fluorescence micrographs of TuJ1‐immunoreactive differentiated neurons co‐infected with DsRed‐tagged WT and CTF TDP‐43 adenoviruses and EGFP‐tagged hPJA1v1 (top row) or hPJA1ΔR (bottom row) adenovirus in the presence of 0.5 μM MG‐132. The nucleus is counterstained with Hoechst 33342. Two types of co‐localization (i.e., perinuclear round fluorescence [arrows] and cytoplasmic amorphous aggregates [arrowheads]), are observed.

Article Snippet: The blotted membrane was blocked with 3% skim milk and incubated overnight with rabbit anti‐FLAG (#7425; Sigma), rabbit anti‐TDP‐43 C‐terminus (405‐414) (#TIP‐TDP09; Cosmo Bio, Tokyo, Japan), rabbit anti‐phosphorylated TDP‐43 (pSer409/S410) (#TIP‐PTD‐P02; Cosmo Bio), rabbit anti‐PJA1 (#17687‐1‐AP; ProteinTech Japan, Tokyo, Japan), rabbit anti‐Myc Tag (#2278; Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐ubiquitin K48 (#05‐1307; Sigma), rabbit anti‐UBE2D2/3 (#HPA003921: Sigma), rabbit anti‐UBE2E3 (#15488‐1‐AP; ProteinTech), rabbit anti‐UBE2K (#11834‐3‐AP; ProteinTech), and mouse monoclonal anti‐GAPDH (#ab8245; Abcam, Cambridge, UK) antibodies at dilutions of 1:1000, followed by incubation with biotinylated anti‐rabbit or anti‐mouse IgG (Vector Laboratories, Burlingame, CA, USA; 1:1000) for 1 h and streptavidin‐alkaline phosphatase (#11089161001; Sigma; 1:1000) for 1 h. Reactions were visualized by color development using nitroblue tetrazolium chloride (NBT) and 5‐bromo‐4‐chloro‐3‐indolylphosphate p‐toluidine salt (BCIP) (#11681451001; Sigma).

Techniques: Binding Assay, Western Blot, Expressing, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Fluorescence