Journal:

Article Title: Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

doi: 10.1182/blood-2006-04-015586

Figure Lengend Snippet: Akt is preferentially activated in the blastoid variant of MCL and in MCL cell lines and is associated with phosphorylation of several downstream targets. (A) Western blot analysis for activated p-Akt (Ser473) in MCL cases and cell lines (Granta 519 [G], NCEB-1 [N], REC-1 [R], Z138C [Z]). High levels of p-Akt were detected in 12 of 12 blastoid MCLs (cases 1-12) and in the 4 cell lines, whereas only 5 of 19 typical MCLs displayed p-Akt—3 at high levels (cases 14, 16, and 18) and 2 at low levels (cases 24 and 27). Actin is shown as loading control. (B) Western blot analyses of downstream Akt targets shown for representative MCL cases, the Granta 519 cell line, and a follicular hyperplasia control (FHP). Blastoid variants (cases 1-4) and the Granta 519 cell line show high levels of p-Akt and multiple phosphorylated downstream targets (p-FRKHL-1, p-MDM2, p-p27kip1, p-Bad), compared with typical MCL (cases 22-24). The low activation of Akt in case 24 results in modest phosphorylation of the downstream targets. Note that GSK-3β, a classic Akt target, is phosphorylated in both p-Akt+ and p-Akt-cases. The negative control follicular hyperplasia (FHP) shows no activation of the Akt pathway. Cyclin D1 is strongly positive in all MCL cases. Actin shows equal loading of protein for each lane.

Article Snippet: For immunoblotting the following antibodies were used: cyclin D1 (DCS6; BD Bioscience, Franklin Lakes, NJ), PTEN (6H2.1; Cascade Bioscience, Winchester, MA), α-tubulin (Sigma Aldrich, St Louis, MI), actin (ACTN05; LabVision, Fremont, CA), Bcl-xL (2H12; Zymed, San Francisco, CA), p-p27 (Thr157) and p27 (kip1) (R&D Systems, Minneapolis MN), p-FKHRL-1 (Ser253) and oct-1 (Santa Cruz Biotechnology, CA); p-Akt (Ser473) and Akt, p-MDM2 (Ser166), Bad, p-Bad (Ser136), p-GSK-3β (Ser9), cleaved caspase 3, p-p65 (Ser536), p65 (NF-κB), p-IκBα (Ser32), p-mTOR (Ser2448), p-p70S6 (Thr389), p-S6K (Ser235/236), anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP)-linked, from Cell Signaling (Beverly, MA).

Techniques: Variant Assay, Phospho-proteomics, Western Blot, Control, Activation Assay, Negative Control

Journal:

Article Title: Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

doi: 10.1182/blood-2006-04-015586

Figure Lengend Snippet: Activation of Akt and downstream targets are dependent upon PI3K activity. PI3K/Akt pathway inhibition studies were performed with all 4 MCL cell lines using 3 different inhibitors: LY294002, wortmannin, and Akt inhibitor (Calbiochem). Comparable results were obtained for each line, and results with Granta 519 using LY294002 or Akt inhibitor are shown in representative experiments. Akt inactivation occurs after 8 hours (8 h), followed by the abrogation of phosphorylation of Bad, FRKHL-1, MDM2, and p27kip1 after 24 hours (24 h). Translational control proteins mTOR, p70S6K, and S6K show a time-dependent decrease in phospho-protein levels. S6K and total Akt levels are shown as loading control.

Article Snippet: For immunoblotting the following antibodies were used: cyclin D1 (DCS6; BD Bioscience, Franklin Lakes, NJ), PTEN (6H2.1; Cascade Bioscience, Winchester, MA), α-tubulin (Sigma Aldrich, St Louis, MI), actin (ACTN05; LabVision, Fremont, CA), Bcl-xL (2H12; Zymed, San Francisco, CA), p-p27 (Thr157) and p27 (kip1) (R&D Systems, Minneapolis MN), p-FKHRL-1 (Ser253) and oct-1 (Santa Cruz Biotechnology, CA); p-Akt (Ser473) and Akt, p-MDM2 (Ser166), Bad, p-Bad (Ser136), p-GSK-3β (Ser9), cleaved caspase 3, p-p65 (Ser536), p65 (NF-κB), p-IκBα (Ser32), p-mTOR (Ser2448), p-p70S6 (Thr389), p-S6K (Ser235/236), anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP)-linked, from Cell Signaling (Beverly, MA).

Techniques: Activation Assay, Activity Assay, Inhibition, Phospho-proteomics, Control

Journal:

Article Title: Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

doi: 10.1182/blood-2006-04-015586

Figure Lengend Snippet: Inhibition of the PI3K/Akt pathway leads to up-regulation of p27kip1 and loss of cyclin D1 expression. PI3K/Akt pathway inhibition studies were performed with the 4 MCL cell lines and the non-Akt-activated control cell line RAJI using 3 different inhibitors: LY294002, wortmannin, and Akt inhibitor (Calbiochem). Comparable results were obtained for each MCL cell line, and results with Granta 519 using LY294002 or Akt inhibitor are shown as representative experiments. (A,C) Proliferation/viability as assessed by MTT test. Results are averages of 3 individual experiments and are displayed as percent absorbance of control cells (untreated) with Granta 519 (□), Z138C (), and RAJI (▪). A significant reduction in absorbance to 50% of control values after 48 hours is seen in the MCL cell lines, whereas the non-Akt-activated control RAJI shows no significant reduction. (B,D) Western blot analysis with Granta 519 demonstrates Akt inactivation at 8 hours, followed by abrogation of phosphorylation of FRKHL-1, p27kip1, and GSK-3β by 24 hours. The cell-cycle inhibitor p27kip1 shows a gradual increase in expression levels over time, and cyclin D1 is dramatically down-regulated. In contrast, cdk4 and cyclin E remain constant. α-tubulin expression is shown as loading control.

Article Snippet: For immunoblotting the following antibodies were used: cyclin D1 (DCS6; BD Bioscience, Franklin Lakes, NJ), PTEN (6H2.1; Cascade Bioscience, Winchester, MA), α-tubulin (Sigma Aldrich, St Louis, MI), actin (ACTN05; LabVision, Fremont, CA), Bcl-xL (2H12; Zymed, San Francisco, CA), p-p27 (Thr157) and p27 (kip1) (R&D Systems, Minneapolis MN), p-FKHRL-1 (Ser253) and oct-1 (Santa Cruz Biotechnology, CA); p-Akt (Ser473) and Akt, p-MDM2 (Ser166), Bad, p-Bad (Ser136), p-GSK-3β (Ser9), cleaved caspase 3, p-p65 (Ser536), p65 (NF-κB), p-IκBα (Ser32), p-mTOR (Ser2448), p-p70S6 (Thr389), p-S6K (Ser235/236), anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP)-linked, from Cell Signaling (Beverly, MA).

Techniques: Inhibition, Expressing, Control, Western Blot, Phospho-proteomics

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression on quiescent prostate cancer cells. (A) Cell surface HER2 expression on PCa cell lines (PC3, DU145, C4-2B, LNCaP) and benign prostate epithelial cells (PNT2) cultured in 10% FBS measured by flow cytometry and mean fluorescence intensity (MFI). Open histograms represent an isotype control antibody. Shaded histograms are a HER2 antibody. The vertical line and arrow represent positive expression as defined by the isotype control antibody. (B) Flow cytometry for surface HER2 and BrdU incorporated into DNA of PC3 cells (C) Histograms showing HER2 cell surface expression on the same cell lines cultured in the presence or absence of FBS. (D) Quantification of HER2 surface expression from panel (B) as defined by mean fluorescence intensity (MFI). (E) Immunofluorescence imaging of PC3 (left) or C4-2B (right) cells cultured in either 10% FBS or serum starved conditions showing endogenous p27 and HER2 expression. HER2 (red), p27 (green) and nuclei (DAPI, blue). Scale bar, 50 μm. (F) Cell surface HER2 as measured by flow cytometry of PCa cells cultured with either 1 µM abemaciclib or 0.01% ethanol vehicle. (G) Western blot for HER2 from indicated cell lines cultured for 2 days with or without FBS. β-actin was a loading control. Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: For p27 labeling,Cells were fixed with cold 70% ethanol and stained with anti-p27 antibody (R&D systems, Cat #: IC2256V) in PBS containing 1% FBS for 30 min at room temperature.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Control, Immunofluorescence, Imaging, Western Blot

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression is associated with p27 expression. Left: PC3, Right: C4-2B. Top: Flow cytometry histograms demonstrating cell surface (labeling before fixation) HER2 expression on PC3 and C4-2B cells. Cells were gated by lowest 10% or highest 10% of HER2 levels (HER2 low and HER2 high) populations. Middle: Example flow cytometry histograms of p27 labeling from either the lowest (blue) or highest (red) 10% of HER2 surface expression. Bottom: Quantified p27 expression in the low vs. higher HER2 populationsp27 levels were measured as mean fluorescence intensity (MFI). Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: For p27 labeling,Cells were fixed with cold 70% ethanol and stained with anti-p27 antibody (R&D systems, Cat #: IC2256V) in PBS containing 1% FBS for 30 min at room temperature.

Techniques: Expressing, Flow Cytometry, Labeling, Fluorescence

Journal: Oncotarget

Article Title: The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

doi: 10.18632/oncotarget.6609

Figure Lengend Snippet: A. Lysates of HeLa cells stably overexpressing B56γ3HA or vector transfected with expression vector of Flag-p27 were analyzed by SDS-PAGE and immunoblotting with antibodies specific for phospho-p27 (Thr187), phospho-p27 (Thr157), phospho-p27 (Thr198), phospho-p27 (Ser10), total p27, HA, B56γ, and β-actin. B. Lysates of HeLa cells with vector only or B56γ3 knockdown transfected with expression vector of Flag-p27 were analyzed as described in (A). C. Lysates of HeLa with vector only, B56γ3 overexpression, or B56γ3 knockdown and D. lysates of HCT116 cells with vector only or B56γ3 overexpression were analyzed for levels of phospho-p27 (Thr157), p27, HA, B56γ3 and β-actin as described above. The amounts of protein applied to the gel were adjusted so that the lysates from different cells yielded similar total p27 levels detected on the blot. The relative expression level of each phospho-p27 were quantified by densitometry and normalized with total Flag-p27 or p27. The data shown are expressed as -fold expression level over that of vector control, which was set as 1. Data shown are from one representative experiment of at least two experiments with similar results.

Article Snippet: Antibodies employed include mouse monoclonal anti-HA tag (6E2), rabbit monoclonal anti-HA tag (C2F9), anti-phospho-Akt (Thr308) (C31E5), anti-phospho-Akt (Thr473) (D9E) and anti-lamin A/C from Cell Signaling; anti-PP2A/A (C-20), anti-CDK2 (M2) and anti-p27 (C19) from Santa Cruz; anti-Akt monoclonal antibody, anti-PP2A/C and anti-p27 from BD Transduction Laboratories; anti-tubulin, clone DM1A, and anti-phospho-Kip1 (Thr187) from Upstate; anti-β-actin and anti-FLAG (M2) from Sigma; anti-HA tag monoclonal antibody (HA.11) from Covance; anti-GST antibody and Glutathione-Sepharose from GE Healthcare; anti-phospho-p27/Kip1 (Thr157) and anti-phospho-p27/Kip1 (Thr198) from R&D Systems; anti-phospho-p27 (Ser10) from Invitrogen.

Techniques: Stable Transfection, Plasmid Preparation, Transfection, Expressing, SDS Page, Western Blot, Knockdown, Over Expression, Control

Journal: Oncotarget

Article Title: The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

doi: 10.18632/oncotarget.6609

Figure Lengend Snippet: A. In vitro dephosphorylation reactions of phospho-p27 in the absence or presence of various amounts of B56γ3-containing PP2A complexes with or without 1 μM okadaic acid (OA) were carried out at 37°C for 30 min according to the procedure described under the “Materials and Methods”. Expression levels of phospho-p27 (Thr157), GST-p27, 4HA-B56γ3, and PP2A A and C subunits were detected by immunoblotting with antibodies specific for phospho-p27 (Thr157), phospho-p27 (Ser10), phospho-p27 (Thr198), GST, HA, PP2A/A and PP2A/C. The levels of p27 phosphorylation were quantified by densitometry and normalized with total p27. Levels of control reactions with no addition of PP2A-B56γ3 complexes were set as 100 %. Data expressed as percentages of reduction of phospho-p27 in individual reactions in the presence of PP2A-B56γ3 complexes with or without OA. Data shown are from one representative experiment of at least two experiments with similar results.

Article Snippet: Antibodies employed include mouse monoclonal anti-HA tag (6E2), rabbit monoclonal anti-HA tag (C2F9), anti-phospho-Akt (Thr308) (C31E5), anti-phospho-Akt (Thr473) (D9E) and anti-lamin A/C from Cell Signaling; anti-PP2A/A (C-20), anti-CDK2 (M2) and anti-p27 (C19) from Santa Cruz; anti-Akt monoclonal antibody, anti-PP2A/C and anti-p27 from BD Transduction Laboratories; anti-tubulin, clone DM1A, and anti-phospho-Kip1 (Thr187) from Upstate; anti-β-actin and anti-FLAG (M2) from Sigma; anti-HA tag monoclonal antibody (HA.11) from Covance; anti-GST antibody and Glutathione-Sepharose from GE Healthcare; anti-phospho-p27/Kip1 (Thr157) and anti-phospho-p27/Kip1 (Thr198) from R&D Systems; anti-phospho-p27 (Ser10) from Invitrogen.

Techniques: In Vitro, De-Phosphorylation Assay, Expressing, Western Blot, Phospho-proteomics, Control

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 4. EXOSC5 Knockdown led to G1 Arrest in GC. (A,B) Flow cytometry showing the percentages of EXOSC5 knockdown cells and control cells at different cell cycle phase. (C) Cell cycle related proteins (P21, P27, Cyclin D1) in EXOSC5 knockdown and EXOSC5 overexpression cells by Western blot. Data are shown as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques: Knockdown, Flow Cytometry, Control, Over Expression, Western Blot, Standard Deviation

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 5. EXOSC5 promote GC growth by AKT and STAT3 activation. (A) KEGG pathway analysis of the genes significantly associated with the EXOSC5 expression in GC from cBioPortal and Coexpedia. (B) The protein expression levels of p-AKT, AKT, p-STAT3 and STAT3 in GC cell lines after EXOSC5 silencing or overexpressing. (C, D) Proliferation ability of MKN45 cells with EXOSC5 overexpression was determined by CCK8 and colony formation assay after treatment with MK-2206 (10 uM), S31-201 (10 uM) and DMSO. (E) The protein expression levels of p-AKT, AKT, p-STAT3, STAT3 and cell cycle related proteins (cyclin D1, p21 and p27) in MKN45 cells with EXOSC5 overexpression after treatment with MK-2206, S31-201 and DMSO. Data are shown as the mean ± standard deviation. ***P < 0.001.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques: Activation Assay, Expressing, Over Expression, Colony Assay, Standard Deviation

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 6. EXOSC5 promote GC growth by AKT and STAT3 activation in GC organoid and vivo. (A) Hematoxylin-eosin staining (H&E) of GC organoid (400x). (B) Immunohistochemistry (IHC) of GC organoid after EXOSC5 silencing. (C) The protein expression levels of p-AKT, AKT, p-STAT3, STAT3 and cell cycle related proteins (cyclin D1, p21 and p27) in GC organoid after EXOSC5 silencing or overexpressing. (D, E) The growth of organoid model with EXOSC5 knockdown or overexpression was assessed every 7 days. (F) Representative images of subcutaneous tumors in nude mice injected HGC27 cells transferred with shRNA. (G, H) Tumor volumes were measured by growth curve every 5 days and final weights of tumor were measured on the terminal days. (I) EXOSC5 staining in xenografted HGC27 tumors silencing EXOSC5. Data are shown as the mean ± standard deviation. **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques: Activation Assay, Staining, Immunohistochemistry, Expressing, Knockdown, Over Expression, Injection, shRNA, Standard Deviation

Journal: Journal of Cancer

Article Title: EXOSC5 promotes proliferation of gastric cancer through regulating AKT/STAT3 signaling pathways.

doi: 10.7150/jca.69166

Figure Lengend Snippet: Figure 7. Schematic model of EXOSC5/AKT/STAT3 axis in GC. EXOSC5 promotes growth and survival in GC by regulating cell cycle proteins (P21, P27, Cyclin D1) via AKT and STAT3 pathways.

Article Snippet: The primary antibodies are as following: EXOSC5 (Proteintech, 15627-1-AP); p21 (Proteintech, 10355-1-AP); p27 (Proteintech, 25614-1-AP); cyclin D1 (CST, 2922S); p-AKT (CST, 9271S); AKT (CST, 9272S); p-STAT3 (CST, 9145); AKT (CST, 9272S).

Techniques:

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 5. Effect of SER5 on HIV-1 particles pseudotyped with different envelopes (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with envelopes of HIV-1 BaL (red), FeLV-A (green), FeLV-B (blue) or amphotropic (ampho) Env (orange) were produced in the presence of increasing amounts of feline SERINC5 (feSER5) or (B) human SERINC5 (huSER5) (0, 100, 200 or 400 ng). Viral infectivity was determined after normalization for reverse transcriptase activity by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001 to 0.01 very significant (**), 0.01 to 0.05 significant (*), >0.05 not significant (ns). (C) SERINC5 stability in the presence of increasing amounts of FeLV-B envelope. HEK293T cells were transfected with fe/huSER5 and increasing amounts of FeLV-B envelope expression plasmids or empty vector as control. Cells were harvested and analyzed by immunoblotting using anti-p85/70, anti-HA and anti-tubulin antibodies. a p85/70 detects envelope (SU-TM p85) and SU only (p70), a HA detects HA-SER5.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Infection, Reverse Transcription, Activity Assay, Luciferase, Transfection, Expressing, Control, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 6. Generation of FeLV glycoGag expression constructs. (A) Sequences of FeLV-A and FeLV-B glycoGags. The highlighted amino acids correspond to disagreements, which are colored according to their side chain chemistry. Cytoplasmic and transmembrane domains are indicated. (B) Plasmids expressing the HA-tagged FeLV glycoGag were produced by amplifying the glycoGag from pFGA-5 and pFGB (FeLV-A and FeLV-B glycoGag respectively), yielding a 9.6 kDa protein. (C) HEK293T cells were transfected with the generated expression plasmids, harvested two days post-transfection and analyzed by immunoblotting using anti-HA and anti-tubulin antibodies.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Expressing, Construct, Produced, Transfection, Generated, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 7. Effect of FeLV glycoGag on HIV-1 in the presence of SER5. (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 BaL envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) together with FeLV-A glycoGag, FeLV-B glycoGag or empty vector. Particles were normalized by RT activity and used for infections. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns). (B) Effect of SERINC and glycoGag on HIV-1 cell-to-cell transmission. HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 envelope BaL were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV-A or -B glycoGag. After transfection, virus producer HEK293T cells were co-cultured with HEK293T transfected with expression plasmids for human CD4 and human CCR5. The infectivity determined by quantification of luciferase activity of a vector that is detectable only after reverse transcription of the spliced luciferase gene. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001– 0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase, Transmission Assay, Transfection, Virus, Cell Culture, Expressing, Reverse Transcription

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 8. Effect of FeLV glycoGag or FeLV-B envelope on FIV in the presence of SER5. FIV particles (3- plasmid system) pseudotyped with FIV EE14 envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV glycoGag or in the absence of FIV EE14 envelope and glycoGag and pseudotyped by FeLV-B envelope. Particles were normalized by RT activity and used for infection. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 9. Comparison of delta Ct values of feSER5 in transfected HEK293T cells and in cat cells and surface expression of feSER5 in the presence of different FeLV proteins. (A) HEK293T cells were transfected with different amounts of feSER5 plasmid. The endogenous feSER5 were evaluated in feline CRFK cell lines and PBMC or macrophages from one cat. (B) HEK293T cells were cotransfected with feSER5-iHA and empty plasmid, Nef, MLV glycoGag , FeLV-A/B glycoGag or FeLV-A/B envelope. The percent of feSER5 positive cells was evaluated by flow cytometry analysis and the total of feSER5 positive cells were scaled to 100%. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01– 0.05 significant (*), >0.05 not significant (ns). (C) Representative plots of one of three experiments showing the surface expression of feSER5-iHA (HA-Alexa Fluor 488). Green fluorescence was quantified using the FITC-A channel vs. FSC-A (10,000 cells were counted).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Comparison, Transfection, Expressing, Plasmid Preparation, Cytometry

Journal: Journal of Cancer

Article Title: Oxymatrine Synergistically Potentiates the Antitumor Effects of Cisplatin in Human Gastric Cancer Cells.

doi: 10.7150/jca.28532

Figure Lengend Snippet: Figure 6. OMT and CDDP act synergistically to inhibit the AKT/ERK pathway. (a) Western blotting assay was used to analysis the expression level of cyclin D1, p21, p27, AKT, p-AKT, ERK and p-ERK. (b) The densitometry analysis of every factor was performed, normalized with the corresponding GAPDH content. *P < 0.01 versus OMT or CDDP alone group.

Article Snippet: AKT, p-AKT, ERK, p-ERK, cyclinD1, p21 and p27 antibodies were purchased from Cell Signaling Technology (MA, USA), and the GAPDH antibody was from Kangchen Bio-tech (Shanghai, China).

Techniques: Western Blot, Expressing