anti-mouse sirt2  (ZenBio)

Journal: Neurochemistry international

Article Title: Proteostasis and autophagy disruption by the aging-related VGVAPG hexapeptide - preliminary insights into a potential novel elastin-induced neurodegeneration pathway in an in vitro human cellular neuron model.

doi: 10.1016/j.neuint.2025.105992

Figure Lengend Snippet: Fig. 2. Interaction between VGVAPG-induced SIRT2 and HRD1 ligase HRD1 mRNA expression (n=3) after the treatment of the differentiated SH-SY5Y cells with 10 nM of VGVAPG, 10 µM of AGK2, or in co-treatment for 24h, (A). HRD1 (B) and SIRT2 (C) protein expression (n=3) after the treatment of the cells with 10 nM of VGVAPG, 10 µM of AGK2, or in the co-treatment for 48h. Immunofluorescence staining of Ac-α-tub (AlexaFluor595, red channel)/nuclei (DAPI, blue channel) after the treatment of the differentiated SH-SY5Y cells with 10 nM of VGVAPG for 48h (D). The main hypothesis tested in this paper is presented as scheme E. Visualization of HRD1 expression using immunofluorescence staining anti-HRD1 ligase (AlexaFluor595; red channel)/nuclei (DAPI; blue channel) after the treatment of the differentiated SH-SY5Y cells with 10 nM of VGVAPG for 48h (F). The inset of LUTs and inverted LUTs are shown next to the original image as well as the HRD1-AlexaFluor594 fluorescence intensity level (n=25). Scale bar = 40 µm, Magnification = 400x (ZEISS Plan-Apochromat 40x/1.3 Oil DIC (UV) VIS-IR objective). Means ± SD (standard deviations) denoted as *** are statistically different at p<0.001, compared to the control.

Article Snippet: For the multi-stained experiment (#IF3), after 266 incubation with the secondary antibodies, the slides were blocked for 30 minutes with 5% 267 goat serum, and primary mouse anti-SIRT2 antibodies (Santa Cruz Biotechnology, cat. sc-268 28298, dilution 1:100) were added.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: Neurochemistry international

Article Title: Proteostasis and autophagy disruption by the aging-related VGVAPG hexapeptide - preliminary insights into a potential novel elastin-induced neurodegeneration pathway in an in vitro human cellular neuron model.

doi: 10.1016/j.neuint.2025.105992

Figure Lengend Snippet: Fig. 5. Inhibitory effect of VGVAPG on UPS leads to SIRT2 accumulation and ER stress. Caspase-like (β1 subunit) – A; trypsin-like (β2 subunit) – B; and chymotrypsin-like (β5 subunit) – C proteasome activities (n = 6), along with the expression of ubiquitinated proteins (n = 3) – D, were measured after treating neurons with 10 nM VGVAPG, 10 µM AGK2 alone, or in the co-treatment for 48 h. A schematic summarizing the VGVAPG-induced interactions between specific intracellular components is shown on the right (E). Representative blots are shown on the left. Means ± SD (standard deviations) denoted as *** are statistically different at p<0.001, compared to the control (ANOVA). The GAPDH protein expression was always used as a loading control.

Article Snippet: For the multi-stained experiment (#IF3), after 266 incubation with the secondary antibodies, the slides were blocked for 30 minutes with 5% 267 goat serum, and primary mouse anti-SIRT2 antibodies (Santa Cruz Biotechnology, cat. sc-268 28298, dilution 1:100) were added.

Techniques: Expressing, Control

Journal: Neurochemistry international

Article Title: Proteostasis and autophagy disruption by the aging-related VGVAPG hexapeptide - preliminary insights into a potential novel elastin-induced neurodegeneration pathway in an in vitro human cellular neuron model.

doi: 10.1016/j.neuint.2025.105992

Figure Lengend Snippet: Fig. 6. VGVAPG-induced autophagy correlates with the SIRT2 overexpression as a result of inhibition of UPS. Immunofluorescence staining (A) of SIRT2 (AlexaFluor594; red channel), ATG18 (FITC; green channel), and nuclei (DAPI; blue channel) in the differentiated SH-SY5Y cells treated with 10 nM of VGVAPG, 1 µM of LS- 102, or after the co-treatment for 24h. Quantification of SIRT2 (n=40) (B) and ATG18 (n=40) (C) fluorescence. The representative insets (1 – 4) are shown below, together with the colocalization analysis and its quantification based on Pearson’s correlation (n=3) (D). The white arrows indicate the obvious colocalization. Scale bar = 50 µm. Magnification = 630× (ZEISS Plan-Apochromat 63×/1.40 Oil DIC M27). Means ± SD (standard deviations) denoted as *** are statistically different at p<0.001, compared to the control, while means denoted as # are statistically different between certain groups at p<0.05 (t-test).

Article Snippet: For the multi-stained experiment (#IF3), after 266 incubation with the secondary antibodies, the slides were blocked for 30 minutes with 5% 267 goat serum, and primary mouse anti-SIRT2 antibodies (Santa Cruz Biotechnology, cat. sc-268 28298, dilution 1:100) were added.

Techniques: Over Expression, Inhibition, Immunofluorescence, Staining, Fluorescence, Control

Journal: Frontiers in Microbiology

Article Title: SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters

doi: 10.3389/fmicb.2022.836446

Figure Lengend Snippet: SIRT2 is correlated with chronic HBV infection. (A,B) The mRNA and protein levels of SIRT2 from PBMC or serum in CHB patients and healthy individuals were analyzed by real-time PCR and ELISA, β-actin was used as an internal control in real-time PCR. (C–E) Correlation between serum SIRT2 level and HBV viral load (C) , HBsAg (D) , HBeAg levels (E) and HBcrAg (F) . HBcrAg level in the serum of CHB patients was detected by fully automated lumipulse chemiluminescence enzyme immunoassay system (Fujirebio Inc., Tokyo, Japan). The HBV viral load, HBsAg and HBeAg levels were log 10 transformed. The correlation co-efficiency ( r ) and two-tailed p values were calculated via Pearson correlation. (G,H) Analysis of correlation between serum SIRT2 level and transaminase ALT, and AST levels using Pearson’s test.

Article Snippet: Rabbit anti-β-actin polyclonal antibody (100 μg/ml; sc-1, 616-R) and mouse anti-SIRT2 monoclonal antibody (200 μg/ml; sc-28, 298) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States), and mouse anti-p53 monoclonal antibody (72 μg/ml; #48818) was obtained from Cell Signaling Technology (United States).

Techniques: Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Transformation Assay, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters

doi: 10.3389/fmicb.2022.836446

Figure Lengend Snippet: Ectopic expression of SIRT2 promotes HBV transcription and replication. HepG2-NTCP cells or PHH were infected with 1 × 10 3 genome equivalents/cells of HBV for 16 h and then transduced with lentivirus expressing SIRT2 isoform 1. Cells were harvested to test the overexpression efficiency and examine the viral markers after 5 days. (A) The overexpression efficiency of SIRT2 was determined by Western blot. (B–D) SIRT2 overexpression elevated total HBV RNAs level and HBV 3.5-kb RNA level, as demonstrated by real-time PCR (B,C) and Northern blot (D) . Each panel was loaded with an equal amount of total RNA and the HBV RNAs were hybridized with DIG-labed single-stranded HBV RNA probe with a length of 1,000 bp. Ribosomal RNAs (28 s and 18 s) served as loading controls. (E,F) SIRT2 overexpression enhanced HBV core DNA level according to real-time PCR (E) and Southern blot (F) . (G,H) HBsAg and HBeAg levels were measured by ELISA. Representative data are from at least three independent experiments. Data was shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit anti-β-actin polyclonal antibody (100 μg/ml; sc-1, 616-R) and mouse anti-SIRT2 monoclonal antibody (200 μg/ml; sc-28, 298) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States), and mouse anti-p53 monoclonal antibody (72 μg/ml; #48818) was obtained from Cell Signaling Technology (United States).

Techniques: Expressing, Infection, Transduction, Over Expression, Western Blot, Real-time Polymerase Chain Reaction, Northern Blot, Southern Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters

doi: 10.3389/fmicb.2022.836446

Figure Lengend Snippet: Gene silencing of SIRT2 inhibits HBV transcription and replication. HepG2-NTCP cells or PHH were infected with 1 × 10 3 genome equivalents/cells of HBV for 16 h and then transduced with lentivirus expressing indicated shRNA (shSIRT2-1 and shSIRT2-2) and scramble control (shCont). Cells were harvested to test the knockdown efficiency and examine the viral markers after 5 days. (A) The efficiency of SIRT2 silencing was examined via Western blot. (B–D) SIRT2 suppression decreased total HBV RNAs level and HBV 3.5-kb RNA level based on real-time PCR (B,C) and Northern blot (D) . Each panel was loaded with an equal amount of total RNA and the HBV RNAs were probed with DIG-labeled single-stranded HBV RNA probe with a length of 1,000 bp. Ribosomal RNAs (28 s and 18 s) were used as loading controls. (E,F) SIRT2 knockdown suppressed HBV core DNA level as determined by real-time PCR (E) and Southern blot (F) . (G,H) HBsAg and HBeAg levels were detected by ELISA. Representative data are from at least three independent experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit anti-β-actin polyclonal antibody (100 μg/ml; sc-1, 616-R) and mouse anti-SIRT2 monoclonal antibody (200 μg/ml; sc-28, 298) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States), and mouse anti-p53 monoclonal antibody (72 μg/ml; #48818) was obtained from Cell Signaling Technology (United States).

Techniques: Infection, Transduction, Expressing, shRNA, Control, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Northern Blot, Labeling, Southern Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters

doi: 10.3389/fmicb.2022.836446

Figure Lengend Snippet: SIRT2 regulates HBV cccDNA transcription activity. (A,B) HepG2-NTCP cells were infected with HBV for 16 h and then transduced with indicated lentivirus for 4 days and treated with Actinomycin D (5 μg/ml). Cells were harvested at indicated time. Total HBV RNAs (A) and HBV 3.5-kb RNA (B) were examined by real-time PCR, and the amount of RNA at time zero was set as 100%. (C,D) HBV-infected HepG2-NTCP cells were incubated with 0.2 mM 5-ethynyl uridine (EU) for 24 h. The newly synthesized EU-labeled RNA was purified from the total RNA, and EU-labeled HBV RNAs were quantified by real-time PCR. (E,F) HBV infected-HepG2-NTCP cells were transduced with indicated lentivirus. 5 days later, HBV cccDNA was extracted with Hirt lysis buffer and analyzed via Taqman-probe specific real-time PCR. The ratios of total RNA/cccDNA and 3.5-kb RNA/cccDNA indicated the transcriptional activity of cccDNA. (G,H) The effect of SIRT2 overexpression or down-regulation on HBV EnI/Xp, EnII/Cp, Sp1 and Sp2 were determined by dual-luciferase reporter assay. The transfection efficiency was normalized under co-transfection with plasmid RL-TK. Representative data are from at least three independent experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit anti-β-actin polyclonal antibody (100 μg/ml; sc-1, 616-R) and mouse anti-SIRT2 monoclonal antibody (200 μg/ml; sc-28, 298) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States), and mouse anti-p53 monoclonal antibody (72 μg/ml; #48818) was obtained from Cell Signaling Technology (United States).

Techniques: Activity Assay, Infection, Transduction, Real-time Polymerase Chain Reaction, Incubation, Synthesized, Labeling, Purification, Lysis, Over Expression, Luciferase, Reporter Assay, Transfection, Cotransfection, Plasmid Preparation

Journal: Frontiers in Microbiology

Article Title: SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters

doi: 10.3389/fmicb.2022.836446

Figure Lengend Snippet: SIRT2 regulates p53 level by targeting the p53 promoter. (A) Real-time PCR analysis of gene expression of various transcription factors associated with HBV transcription. (B) HepG2-NTCP and PHH cells were transduced with lentivirus expressing shSIRT2-1/shSIRT2-2 and shCont. The effect of SIRT2 knockdown on p53 mRNA level was analyzed by real-time PCR. (C,D) The effect of SIRT2 overexpression or SIRT2 depletion on p53 protein level in HepG2-NTCP and PHH cells was examined by Western blot. β-actin served as the loading control. (E,F) HepG2-NTCP and Huh-7 cells were transfected with pGL3-p53 promoter and transfected with plasmids expressing SIRT2 or transduced with indicated lentivirus expressing shSIRT2. The luciferase activity was measured at 36 h post transfection using dual-luciferase reporter assay. Representative data are from at least three independent experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit anti-β-actin polyclonal antibody (100 μg/ml; sc-1, 616-R) and mouse anti-SIRT2 monoclonal antibody (200 μg/ml; sc-28, 298) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States), and mouse anti-p53 monoclonal antibody (72 μg/ml; #48818) was obtained from Cell Signaling Technology (United States).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Transduction, Expressing, Knockdown, Over Expression, Western Blot, Control, Transfection, Luciferase, Activity Assay, Reporter Assay

Journal: Frontiers in Microbiology

Article Title: SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters

doi: 10.3389/fmicb.2022.836446

Figure Lengend Snippet: SIRT2 facilitates HBV transcription and replication via repressing the binding of p53 on HBV EnI/Xp and EnII/Cp. (A,B) Effect of SIRT2 overexpression (A) and down-regulation (B) on the recruitment of p53 to HBV EnI/Xp and EnII/Cp was detected by ChIP assay. Cross-linked chromatin from HBV-infected HepG2-NTCP cells was immunoprecipitated with anti-p53 antibody followed by real-time PCR with specific HBV EnI/Xp and EnII/Cp primers. The promoter of GAPDH and MYH6 were used as internal controls. The ChIP results are expressed as % of input. (C) HepG2-NTCP cells were transfected with indicated plasmids. Dual-luciferase assay was performed to examine the activities of HBV EnI/Xp and EnII/Cp. pGL3-EnI/Xp Mut and pGL3-EnII/Cp Mut denoted the p53 binding sites in HBV EnI/Xp and EnII/Cp were mutated. (D–F) HepG2-NTCP cells were infected with HBV WT or HBV Mut (binding sites of p53 to EnI/Xp and EnII/Cp were mutated) and transduced with lentivirus expressing SIRT2. Total HBV RNAs and HBV 3.5-kb RNA levels were analyzed by real-time PCR (D) and Northern blot (E) , HBV core DNA level in HepG2-NTCP cells was analyzed by real-time PCR and Southern blot (F) . (G) HepG2-NTCP cells were transfected with indicated plasmids. Dual-luciferase assay was performed to examine the activities of HBV EnI/Xp and EnII/Cp. (H–J) HBV-infected HepG2-NTCP cells were transfected with plasmids containing SIRT2 or p53. Total HBV RNA and HBV 3.5-kb RNA levels were detected by real-time PCR (H) and Northern blot (I) . HBV core DNA level was measured by real-time PCR and Southern blot (J) . Representative data are from at least three independent experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit anti-β-actin polyclonal antibody (100 μg/ml; sc-1, 616-R) and mouse anti-SIRT2 monoclonal antibody (200 μg/ml; sc-28, 298) were purchased from Santa Cruz Biotechnology (Dallas, TX, United States), and mouse anti-p53 monoclonal antibody (72 μg/ml; #48818) was obtained from Cell Signaling Technology (United States).

Techniques: Binding Assay, Over Expression, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Transduction, Expressing, Northern Blot, Southern Blot

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: HRD1 deficiency upregulated the expression of SIRT2 as determined using mass spectrometry analysis. (A) The network view of the predicted E3 ligase of SIRT2 by UbiBrowser. The query substrate is located in the center of the canvas. The predicted E3 ligases surround the substrate. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. Node size, edge color depth, and edge width are proportional to the confidence score. H, HECT; U, UBOX; F, F-box; R, RING; C, CDC20; D, DWD; S, SOCS. (B and C) Proteomic analysis of genes differentially expressed between control and HRD1 knockout A549 cells. Diagrams depict 87 HRD1-repressed genes and 126 HRD1-activated genes. (D) Representative showing of HRD1-activated and -repressed genes by KEGG analysis.

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Expressing, Mass Spectrometry, Knock-Out

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: SIRT2 interacts with HRD1. (A) The Myc-SIRT2 expression plasmid was cotransfected with or without HRD1-Flag into HEK293T cells. The SIRT2 protein was immunoprecipitated (IP) with the anti-Flag antibody, and the expression of SIRT2 and HRD1 in the whole-cell lysate (WCL) was confirmed by immunoblotting (IB) with antibodies against Myc and Flag. (B) The HRD1-Myc expression plasmid was cotransfected with or without Flag-SIRT2 into HEK293T cells. The HRD1 protein was immunoprecipitated with anti-Flag antibody, and the expression of SIRT2 and HRD1 in the WCL was confirmed by IB with antibodies against Myc and Flag. (C) HEK293T cells were cotransfected with the HRD1-Myc expression plasmid and Flag-SIRT1 to Flag-SIRT7. The HRD1 protein was immunoprecipitated with the anti-Flag antibody. The expression of HRD1 and SIRTs in the WCL was confirmed by IB with antibodies against Myc and Flag. (D) The endogenous interaction of HRD1 and SIRT2 was evaluated in A549 cells. A normal mouse IgG (mIgG) was used as a control. (E) Schematic representation of SIRT2 and its mutants, indicating that SIRT2 contains a CORE domain. (F) Schematic representation of HRD1 and its mutants, showing that HRD1 protein contains an N-terminal signal peptide, transmembrane domain, zinc finger domain, and C-terminal proline-rich domain. (G) The interactions of SIRT2 and its mutants with HRD1 protein were evaluated. HRD1-Flag plasmids were cotransfected with SIRT2 or each of its mutants, and their interactions were analyzed as for panel A. (H) The interactions of SIRT2 with HRD1 and its mutants were evaluated. Flag-SIRT2 plasmids were cotransfected with HRD1 or each of its mutants into HEK293T cells, and their interactions were analyzed as for panel A. For panels A to D, F, and H, numbers on the left are molecular masses, in kilodaltons. (I) Colocalization of HRD1 and PDI, SIRT2 and PDI1, and HRD1 and SIRT2 in HeLa cells. The cellular localization of HRD1, SIRT2, and PDI was examined by immunofluorescence staining with corresponding antibodies. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the DNA.

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: HRD1 promotes SIRT2 ubiquitination. (A) HA-ubiquitin, Flag-SIRT2, and HRD1-Myc plasmids were cotransfected into HEK293T cells. SIRT2 ubiquitination was detected by immunoprecipitation of SIRT2 with the anti-Flag antibody and Western blotting with anti-HA antibody. The protein expression levels of HA-ubiquitin, SIRT2, and HRD1 in the whole-cell lysates were confirmed. (B) HA-ubiquitin and Flag-SIRT2 expression plasmids were cotransfected with HRD1-Myc or Myc-UHRF1 into HEK293T cells. SIRT2 ubiquitination was analyzed as for panel A. (C) Schematic representation of HRD1 and its point mutants. The conserved cysteine (C) residues in the RING domain were replaced with alanine (A). (D) HA-ubiquitin and Flag-SIRT2 expression plasmids were cotransfected into HEK293T cells with HRD1-Myc or with HRD1/CA mutants. The effects of HRD1 and its mutants on SIRT2 ubiquitination were analyzed as for panel A. (E) The interactions of SIRT2 with wild-type (wt) HRD1 or its mutants were analyzed as indicated for panel C. (F) HA-ubiquitin expression plasmids were transfected into the control and the stable knockdown HRD1 A549 cancer cells. SIRT2 ubiquitination was detected via immunoprecipitation of SIRT2 with anti-SIRT2 antibody and Western blotting with anti-UB antibody. The protein expression levels of SIRT2 and HRD1 in the whole-cell lysates were confirmed.

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Immunoprecipitation, Western Blot, Expressing, Transfection

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: HRD1 negatively regulates SIRT2 protein stability. (A and B) HRD1 expression plasmids or empty vectors were cotransfected with Flag-SIRT2 into HEK293T cells. The transfected cells were treated with cycloheximide (CHX) for different times. The protein levels in the treated cells were determined by Western blotting using anti-Flag (A, top) and anti-Myc (A, middle) antibodies (Abs). β-Actin was used as a loading control (A, bottom). The band intensities of SIRT2 proteins were quantified, and their relative levels are shown in panel B. (C and D) HRD1 expression plasmids or empty vectors were transfected into HEK293T cells. The transfected cells were treated with CHX for different times. The protein levels in the treated cells were determined through Western blotting using anti-SIRT2 (C, top) and anti-Myc (C, middle) Abs. β-Actin was used as a loading control (C, bottom). The band intensities of SIRT2 proteins were quantified, and their relative levels are shown in panel D. (E) A fraction of cells from panel A was prepared in parallel for total RNA extraction. The mRNA levels of both SIRT2 and HRD1 were determined by real-time PCR. Their relative levels are indicated. The error bar represents the SEMs from triplicate experiments. **, P < 0.05, two-tailed Student’s t test. (F and G) HRD1 or HRD1/CA mutant plasmids were transfected into HEK293T cells. SIRT2 protein stabilities in the transiently transfected HEK293T cells were examined as described for panels A and B. (H and I) A549 cells stably expressing control shRNA or HRD1-specific shRNA (shHRD1-1 and shHRD1-2) were treated with CHX for the indicated time. SIRT2 protein stabilities were analyzed as described for panel A (H, top). The expression levels of HRD1 (H, middle) were confirmed through Western blotting using β-actin as a loading control (H, bottom). The band intensities of SIRT2 proteins were quantified, and their relative levels are shown in panel I. (J) A549 cells stably expressing control or HRD1 knockdown plasmids were treated with the proteasome inhibitor MG132. The protein levels of SIRT2 (top) and HRD1 (middle) were determined by Western blotting with GAPDH as a loading control (bottom).

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Expressing, Transfection, Western Blot, RNA Extraction, Real-time Polymerase Chain Reaction, Two Tailed Test, Mutagenesis, Stable Transfection, shRNA

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: HRD1 knockdown suppresses lung cancer cell proliferation and tumorigenesis. (A and B) The cell proliferation of A549 cancer cells stably expressing the indicated plasmids or combination of plasmids was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The error bars represent the SEMs from triplicate experiments. HRD1 2CA mutant C291, 294A was used. The protein levels of SIRT2, HRD1, or both were determined by Western blotting using β-actin as a loading control in A549 cells stably expressing control vector or indicated overexpression or knockdown plasmids. (C to F) A clonogenic assay was performed to measure the colony formation capacity of A549 cancer cells stably expressing the indicated plasmids (C) or stably expressing the indicated knockdown plasmids (E). The quantitation of colony number is shown in panels D and F. Error bars represent the SD from triplicate experiments. *, P < 0.05. (G to I) A549 cells stably expressing control, HRD1 knockdown, and HRD1 plasmids were injected subcutaneously into nude mice. Six weeks after injection, tumors were isolated and photographed (G). The tumor sizes were measured and are depicted as tumor weight (H) and tumor volume (I).

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, MTS Assay, Mutagenesis, Western Blot, Plasmid Preparation, Over Expression, Clonogenic Assay, Quantitation Assay, Injection, Isolation

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: HRD1 knockdown inhibits cell migration and invasion in lung cancer cells. (A to D) Wound-healing assays were performed to measure the migration of A549 cells stably expressing the indicated knockdown or overexpression plasmids or a combination of plasmids. Cells were monitored within 24 h to evaluate the rate of migration into the scratched area. The wound edges are indicated by black lines in panels A and C. The relative distances of migration are shown in panels B and D. The results represent the means ± SEMs from triplicate experiments. *, P < 0.05; **, P < 0.01. (E to J) The effects of stable knockdown (E to H) or overexpression (I and J) of HRD1 or SIRT2 on cell invasion were measured in A549 cells (E, F, I, and J) or H446 cells (G and H) by Matrigel Transwell assays. The quantitative results are shown. The results represent the means and SD from triplicate experiments. *, P < 0.05; **, P < 0.01. (K) The protein levels of HRD1, SIRT2, ac-H4K16 were determined by Western blotting using β-actin as a loading control in A549 cells stably expressing control or HRD1 knockdown plasmids.

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Migration, Stable Transfection, Expressing, Over Expression, Western Blot

Journal: Molecular and Cellular Biology

Article Title: E3 Ubiquitin Ligase HRD1 Promotes Lung Tumorigenesis by Promoting Sirtuin 2 Ubiquitination and Degradation

doi: 10.1128/MCB.00257-19

Figure Lengend Snippet: HRD1 and SIRT2 expression in lung cancer. (A) The protein levels of HRD1 and SIRT2 were measured in lung cancer tissue specimens (T; n = 8) and matched with adjacent normal tissues (N; n = 8) through Western blotting. (B) The relative HRD1 and SIRT2 protein levels in normal and cancer tissues were quantified. (C) Representative images showing the immunohistochemical staining of HRD1 and SIRT2 in normal and lung cancer tissues. (D) Analysis of the GEPIA data indicates a significant positive correlation of SIRT2 expression and a negative correlation of HRD1 expression between their expression and the overall survival of lung adenocarcinoma patients. GEPIA uses the log rank test, also known as the Mantel-Cox test, for the hypothesis test. The Cox proportional hazard ratio and the 95% confidence interval information are also included in the survival plot. (E) Schematic model of SIRT2 regulation by HRD1. Upregulation of HRD1 in lung cancer patients leads to downregulation of SIRT2, thus resulting in enhanced cell proliferation and lung tumorigenesis.

Article Snippet: The following antibodies were used: anti-mouse SIRT2 (Zenbio, Chengdu, China), anti-rabbit actin (Sigma-Aldrich), anti-rabbit HRD1 (Sigma-Aldrich), anti-mouse Flag (Sigma-Aldrich), anti-mouse Myc (9E10; Santa Cruz Biotechnology), anti-rabbit ubiquitin (Ub) (Millipore), anti-rabbit HA (Santa Cruz Biotechnology), anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Beyotime, Shanghai, China), anti-rabbit histone H4 (acetyl K16; Zenbio, Chengdu, China), anti-rabbit HIST4H4 (Sangon, Shanghai, China), and anti-mouse antitubulin (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining