Journal: PLoS Pathogens

Article Title: A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis

doi: 10.1371/journal.ppat.1010141

Figure Lengend Snippet: The A549 cells and IGDCC4-KO cells infected with VSV-GFP at an MOI of 10 were incubated and washed differently before being collected for detecting viral attachment ( A ) or ( B ) internalization by using qRT-PCR, the values of IGDCC4-KO cells were normalized with that of A549 cells. Data shown are from three replicates (means ± SDs). ( C ) and ( D ) Internalization of transferrin in IGDCC4-KO and A549 cells were evaluated by incubating cells with 50 μg/ml fluorescently labeled transferrin at 37°C for 30 min before receiving an acid wash to quench extracellular transferrin and were subsequently processed for confocal microscopy. Automatic image analysis quantified a total of 100 cells in three independent experiments and normalized to the A549 cells. The data shown in A, B, and D are from three independent experiments or biological replicates (means ± SDs).

Article Snippet: After incubation for 1 h at 37°C, the cells were treated with 50 μg/ml Alexa 488-labeled transferrin (Thermo Scientific, Cat.no.

Techniques: Infection, Incubation, Quantitative RT-PCR, Labeling, Confocal Microscopy

Journal: International journal of oncology

Article Title: TIP60 governs the auto‑ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex.

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: Figure 1. TIP60 and ubiquitin co‑transfection induces the downregulation of UHRF1. Cells were co‑transfected with either TIP60‑eGFP (green) and RFP‑Ubiquitin (red) or eGFP and RFP‑Ubiquitin. Immunostaining of UHRF1 in HeLa cells without (A) or with treatment by MG‑132 (B). Cells were fixed following transfection and labeled with anti‑UHRF1 antibody. Endogenous UHRF1 protein was labeled with Alexa 647‑labeled secondary antibody before visualization with confocal microscopy. Scale bar, 10 µm. (C and D) Represent mean fluorescence intensities levels of UHRF1 in the different samples. Values are the mean ± SEM for three independent experiments; *P<0.05; ****P<0.0001 (vs. control group), determined by one‑way ANOVA with Tukey's post hoc test. UHRF1, ubiquitin‑like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa.

Article Snippet: Other antibodies used included rabbit polyclonal anti‐HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti‐DNMT1 (1:5,000; cat. no. PTG‐MAB0079, ProteoGenix), mouse monoclonal anti‐ubiquitin (1:500; cat. no. 05‐944, Sigma‐Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002‐1‐Ig, Proteintech Group, Inc.; and cat. no. A‐11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti‐GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti‐GFP (1:1,000; cat. no. 66002‐1‐Ig, Proteintech Group, Inc.), mouse mono‐ clonal anti‐p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti‐caspase‐3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti‐BCL2 (1:1,000; cat. no. 05‐826, Merck KGaA), mouse monoclonal anti‐poly(ADP‐ribose) polymerase (PARP; 1:1,000; cat. no. 51‐6639GR, BD Biosciences) and rabbit polyclonal anti‐BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Ubiquitin Proteomics, Immunostaining, Transfection, Labeling, Confocal Microscopy, Fluorescence, Control

Journal: International journal of oncology

Article Title: TIP60 governs the auto‑ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex.

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: Figure 3. TIP60 induces auto‑ubiquitination of UHRF1 in HeLa cells. Cells stably expressing either UHRF1 WT or UHRF1 C724A‑H741A mutant were transfected with either TIP60-eGFP WT or TIP60ΔMYST‑eGFP mutant. All samples were treated with 10 µM of MG‑132, 8 h before harvesting the cells. Whole cell lysates and immunoprecipitated samples were analyzed by SDS‑PAGE and then immunoblotted with anti‑GFP and anti‑Ubiquitin antibodies. Inputs and IP gels were processed in parallel under similar conditions. UHRF1, ubiquitin‑like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa.

Article Snippet: Other antibodies used included rabbit polyclonal anti‐HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti‐DNMT1 (1:5,000; cat. no. PTG‐MAB0079, ProteoGenix), mouse monoclonal anti‐ubiquitin (1:500; cat. no. 05‐944, Sigma‐Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002‐1‐Ig, Proteintech Group, Inc.; and cat. no. A‐11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti‐GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti‐GFP (1:1,000; cat. no. 66002‐1‐Ig, Proteintech Group, Inc.), mouse mono‐ clonal anti‐p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti‐caspase‐3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti‐BCL2 (1:1,000; cat. no. 05‐826, Merck KGaA), mouse monoclonal anti‐poly(ADP‐ribose) polymerase (PARP; 1:1,000; cat. no. 51‐6639GR, BD Biosciences) and rabbit polyclonal anti‐BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Stable Transfection, Expressing, Mutagenesis, Transfection, Immunoprecipitation

Journal: Neurobiology of disease

Article Title: Inhibition of GSK-3 ameliorates the pathogenesis of Huntington's disease.

doi: 10.1016/j.nbd.2021.105336

Figure Lengend Snippet: Fig. 1. Amounts of mHtt aggregates are regulated by the autophagy- lysosome pathway. A. SH-SY5Y cells were transfected with GFP-Q23 or GFP-mHtt plasmids, and cells expressing GFP-mHtt were starved for amino acids (−aa, 6 h), or treated with vinblastine (20 nM, 6 h). Fixed cells were stained with DAPI and visualized by fluorescence microscopy (left panel). The percentage of transfected cells containing mHtt aggregates is shown at the middle panel. Numbers of mHtt aggregates/cell are shown in Fig. S1. Western blot analysis of cells expressing GFP-mHtt that were starved or not are shown in the right panel showing mHtt, phospho-S6K-1 (Thr389), and autophagic markers. Densitometry analysis of mHtt is show at the right. B. MEF cells (WT) or TSC1/2−/−cells were transfected with GFP-mHtt plasmid, and TSC1/2−/−cells were also subjected to aa starvation. Fixed cells stained with DAPI were visualized by fluorescence microscopy. Numbers of mHtt aggregates/cell is presented in the top panel. Representative Western blot analysis of WT and TSC1/2−/−cells showing mHtt, TSC2, phospho-mTOR (Ser2448), and autophagy markers are shown in the right panel. Densitometry analysis of mHtt is shown in the bottom. C. MEF cells (WT) or ATG5−/−cells were transfected with mCherry-mHtt. Fixed cells stained with DAPI were visualized by fluorescence microscopy. The percentage of transfected cells containing mHtt aggregates is shown in the bottom panel. Western blot analysis of WT and ATG5−/−cells showing mHtt and autophagic/lysosomal markers is shown in the right panel D. SH-SY5Y cells were co-transfected with mCherry-mHtt and GFP-LAMP2 plasmids, and live cells were imaged by confocal microscopy. White square shows magnification of mHtt in lysosome. Treatment with CQ (30 μM, 4 h) is shown at the right end panel. E. Lysosomes were isolated by density-gradient ultracentrifugation from SH-SY5Y cells expressing GFP-mHtt. The lysosome enriched fraction (fraction 1) and the subsequent fractions were immunoblotted for mHtt and CatD. ‘Total’ refers to preparation before organelle fractionation. F. WT and TSC1/2−/−cells were co-transfected with mCherry-mHtt and GFP-LAMP2 plasmids and imaged by confocal microscopy. It is shown that the major portion of mHtt is not localized within lysosomes in the TSC1/2−/−cells. For all panels, gels and data represent 3 independent experiments and results are mean ± SEM, *p < 0.05, ****p < 0.0001, by two-tailed student’s t-test.

Article Snippet: The following antibodies were used: anti-phopsho-S6K-1 (Thr389) (cat#9205), anti-phopsho-mTOr (Ser 2448) (cat#5536), anti-S6 (cat#2217), anti-LC3 (cat#4108), anti-GAPDH (cat#2118), and anti- phospho-AKT (Ser473) (cat#9271) were from Cell Signaling Technologies (Beverly, MA, USA).

Techniques: Transfection, Expressing, Staining, Fluorescence, Microscopy, Western Blot, Plasmid Preparation, Confocal Microscopy, Isolation, Fractionation, Two Tailed Test

Journal: Nature Communications

Article Title: Internalized TSH receptors en route to the TGN induce local G s -protein signaling and gene transcription

doi: 10.1038/s41467-017-00357-2

Figure Lengend Snippet: PKA II activation and localization at the Golgi/TGN are required for efficient TSH-dependent CREB phosphorylation a Subcellular localization of PKA subunits in primary mouse thyroid cells. The catalytic (Cα) and regulatory subunits (RIα, RIIα and RIIβ) of PKA were visualized by immunofluorescence with specific antibodies. Nuclei were stained with DAPI. b , c Co-staining of RIIβ with antibodies against GOLPH4 ( b ) and TGN46 ( c ), showing selective localization of RIIβ subunit on membranes of Golgi and TGN, respectively. Left, C onfocal images. Right, 3D reconstructions from confocal image stacks (see Supplementary Fig. ). d Co-staining of RIIα with GOLPH4, showing preferential localization of RIIα at the Golgi. e Western blot analysis of CREB phosphorylation (Ser133) in response to TSH in cells pretreated for 1 h with a PKA I (Rp8-Br-cAMPS, 250 μM) or PKA II (Rp8-Br-PIP-cAMPS, 250 μM) inhibitor. α-tubulin was used as loading control. Data (mean ± S.E.M, n = 3) are expressed relative to those in the mock-stimulated sample pretreated with PKA I inhibitor. f Induction of early genes in response to TSH in cells pretreated for 1 h with PKA I/II inhibitors. Data (mean ± S.E.M, n = 4) are expressed as in e . g Effect of Ht31 or Ht31P (control) peptides (20 μM, 1 h) on the Golgi localization of RIIβ. Shown are representative RIIβ and GOLPH4 immunofluorescence stainings acquired with same contrast settings. h Quantification of images as in g . RIIβ intensity values (mean ± s.e.m., n = 12/20) are expressed relative to those of GOLPH4. i Western blot analysis of CREB phosphorylation (Ser133) in cells pretreated for 1 h with Ht31 or Ht31P (20 μM) and stimulated with TSH. Data (mean ± s.e.m., n = 3) are expressed relative to those measured in the mock-stimulated control sample. Differences in e , f and i are statistically significant by two-way ANOVA. * P < 0.05, ** P < 0.01, by Bonferroni’s post hoc test. Difference in h is statistically significant by unpaired t -test (*** P < 0.001). ns, statistically non-significant difference. Images in a – d and g are representative of 4, 3, 3, 5 and 3 independent experiments, respectively

Article Snippet: Anti-phospho-CREB (Ser133) rabbit monoclonal antibody (cat. no. 4276; 1:1,000) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Immunofluorescence, Staining, Western Blot, Control

Journal: Nature Communications

Article Title: Internalized TSH receptors en route to the TGN induce local G s -protein signaling and gene transcription

doi: 10.1038/s41467-017-00357-2

Figure Lengend Snippet: TSHR internalization is required for TSH-dependent induction of CREB phosphorylation and gene transcription. a Western blot analysis of CREB phosphorylation (Ser133) in primary mouse thyroid cells stimulated with TSH with/without dynasore pretreatment (30 min). α-tubulin was used as loading control. b Induction of early genes in response to TSH in primary thyroid cells with/without dynasore pretreatment (30 min). Data in a and b (mean ± s.e.m.; n = 5 each) are expressed relative to those measured in mock-stimulated control. Differences in a and b are statistically significant by two-way ANOVA. * P < 0.05 and *** P < 0.001 by Bonferroni’s post hoc test

Article Snippet: Anti-phospho-CREB (Ser133) rabbit monoclonal antibody (cat. no. 4276; 1:1,000) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Nature Communications

Article Title: Internalized TSH receptors en route to the TGN induce local G s -protein signaling and gene transcription

doi: 10.1038/s41467-017-00357-2

Figure Lengend Snippet: Interfering with retrograde trafficking or Golgi/TGN organization impairs cAMP/PKA signaling and CREB phosphorylation. a Sequential HILO images showing effect of BFA on TGN organization and TSH/TSHR retrograde trafficking. Primary mouse thyroid cells were transfected with the TGN maker ST-RFP. Thereafter, they were treated with BFA for 30 min, stimulated with TSH-647 for 10 min and imaged for additional 30 min. b Immunofluorescence against RIIβ and either GOLPH4 (left) or TGN46 (right) in cells pretreated with BFA, showing displacement of the PKA RIIβ subunit from the Golgi and its reduced association with the TGN, respectively. Images were acquired by confocal microscopy. Data in a and b are representative of 3 independent experiments. c cAMP FRET responses (mean ± s.e.m.; n = 9/13) in primary thyroid cells isolated from Epac1-camps mice pretreated with or without (control) BFA for 30 min. d PKA FRET responses (mean ± s.e.m.; n = 11/11) in primary thyroid cells transfected with the AKAR2 sensor and pretreated with or without (control) BFA. Data in c and d were normalized as in Fig. . Differences at individual time points were compared using the Holm–Šídák test for multiple comparisons (black dots, P values). e Western blot analysis of CREB (Ser133) phosphorylation in primary thyroid cells stimulated with TSH with/without BFA pretreatment. α-tubulin was used as loading control. Images are representative of 3 independent experiments. Data (mean ± s.e.m., n = 3) are expressed relative to those measured in the mock-stimulated control sample. f Western blot analysis of CREB (Ser133) phosphorylation in primary thyroid cells transfected with Vps35 siRNA pool or control non-targeting siRNA and stimulated with TSH for 10 min. α-tubulin was used as loading control. Images are representative of six independent experiments. Data (mean ± s.e.m., n = 6) are expressed relative to those measured in the mock-stimulated control sample. Differences in e and f are statistically significant by two-way ANOVA. * P < 0.05 by Bonferroni’s post hoc test. g Schematic view of the results

Article Snippet: Anti-phospho-CREB (Ser133) rabbit monoclonal antibody (cat. no. 4276; 1:1,000) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Control, Western Blot

Journal: Oncology letters

Article Title: Corosolic acid impairs human lung adenocarcinoma A549 cells proliferation by inhibiting cell migration.

doi: 10.3892/ol.2019.10262

Figure Lengend Snippet: Figure 2. CA inhibits VEGFR2 kinase activity. (A) A549 cells were treated with beads only (control) or increasing concentrations of CA (0, 4 and 8 µM) for 24 h, prior to co‑IP with anti‑VEGFR2 antibody, and subsequent western blotting with anti‑phospho‑Serine antibody. Total VEGFR2 and GAPDH were detected in lysates (Input). (B) Quantification of phosphor‑VEGFR2 level shown in (A), normalized by total VEGFR2. (C) CA effects on VEGFR2 kinase activity measured by ADP‑Glo™ kinase assay (n=4; kinase activity data were normalized to the control group and the results are shown as percentages). (D) A549 cell were treated with SU1498 for 24 h and with indicated concentrations of CA for another 24 h. Cell migration ability was measured by Transwell assay. n=4. *P<0.05 and ***P<0.001, compared with the 0 µM group. CA, corosolic acid; VEGRF2, vascular endothelial growth factor receptor 2; IP, immunoprecipitation.

Article Snippet: The rabbit primary antibodies against GAPDH (cat. no. 2118; 1:5,000), VEGFR2 (cat. no. 2479; 1:2,000) and phosphorylated (p)-VEGFR2 (Tyr1059; cat. no. 3817; 1:1,000) were purchased from Cell Signaling Technology (Cell Signaling Technology Europe, B.V., Leiden, The Netherlands).

Techniques: Activity Assay, Control, Western Blot, Kinase Assay, Migration, Transwell Assay, Immunoprecipitation

Journal: Oncology letters

Article Title: Corosolic acid impairs human lung adenocarcinoma A549 cells proliferation by inhibiting cell migration.

doi: 10.3892/ol.2019.10262

Figure Lengend Snippet: Figure 3. CA inhibits VEGFR2‑associated signaling pathway and disrupts cytoskeleton rearrangement. (A) A549 cells were treated with DMSO (control) or indicated concentration of CA for 24 h, prior to assessing VEGFR2 phosphorylation level (Tyr1059) by western blotting. (B) Quantification of WB bands shown in (A). (C) A549 cells were treated with control or indicated concentration of CA for 24 h, prior to assessing Rac1‑GTPase by glutathione‑S‑transferase pull‑down and subsequent western blotting. (D) Quantification of phosphor‑VEGFR2 level (Tyr 1059) shown in (C), normalized by total VEGFR2. n=4. **P<0.01 and ***P<0.001compared with the 0 µM group. (E) A549 cell were treated with DMSO or CA for 24 h and live cells were stained with Tubulin Tracker™ and Mitotracker® for 5 min. Cells were immediately observed by confocal microscopy (magnification, x60; scale bar, 10 µm). CA, corosolic acid; Rac1, Ras‑related C3 botulinum toxin substrate 1; VEGRF2, vascular endothelial growth factor receptor 2; p, phosphorylated.

Article Snippet: The rabbit primary antibodies against GAPDH (cat. no. 2118; 1:5,000), VEGFR2 (cat. no. 2479; 1:2,000) and phosphorylated (p)-VEGFR2 (Tyr1059; cat. no. 3817; 1:1,000) were purchased from Cell Signaling Technology (Cell Signaling Technology Europe, B.V., Leiden, The Netherlands).

Techniques: Control, Concentration Assay, Phospho-proteomics, Western Blot, Staining, Confocal Microscopy

Journal: BMC Neuroscience

Article Title: PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons

doi: 10.1186/s12868-015-0152-8

Figure Lengend Snippet: Up-regulation of PAT1 in primary neurons results in a strong colocalization with APP in the cytoplasm but not at the cell surface. A-B. PAT1-myc was overexpressed by transfection in primary neurons at 5 DIV. 24 h later cells were fixed by PFA 4% only (A) or followed by 0.2% Triton X 100 (B) . Immunolabeling for PAT1-myc (Alexa-488) and endogenous APP (Cy3) was performed and analyzed by confocal microscopy. Two representative images in each condition are presented. In (A) immunolabeling was performed using the anti-myc tag polyclonal #06-549 and the anti-APP-Nter A4 antibody. In (B) the anti-myc tag MABE282 and the anti-APP-Cter polyclonal antibodies were used for immunolabeling. Quantification of colocalization using Volocity software was expressed by Pearson’s coefficient (upper panel). Data presented are the mean ± SEM of 3 independent experiments. Quantification of total APP in conditions of 0.2% Triton X 100 was performed in 23 transfected and 42 non-transfected cells (Ctrl) out of 3 independent experiments. Data are expressed in integrated density / cell in arbitrary units (AU) and represents the mean ± SEM (lower panel). Scale bar: 10 μm. C) Cell surface biotinylation was performed on PAT1-myc overexpressed cells comparatively to control cells (Ctrl). Experiments were performed on 10.10 6 cells 24 h after transfection. NCAM was used as internal control of membrane loading for cell surface biotinylation. The level of APP in biotinylated membranes was reported to NCAM and expressed in arbitrary units (AU). Data are the mean ± SEM of 3 independent experiments. Western blotting were performed using the anti-APP-Nter A4 and the anti-NCAM antibodies.

Article Snippet: Secondary antibodies used for immunocytochemistry were Donkey anti-rabbit cy3 (Cat# 711-166-152, Jackson Immuno-research, Interchim, Montluçon, France) diluted to 1/500, Donkey anti-mouse cy3 (Cat# 715-166-151 Jackson Immuno-research, Interchim), diluted to 1/200 and Donkey anti-mouse Alexa 488 (Cat# 715-546-151 Jackson Immuno-research, Interchim) diluted to 1/200.

Techniques: Transfection, Immunolabeling, Confocal Microscopy, Software, Control, Membrane, Western Blot

Journal: Scientific Reports

Article Title: Induction of MET Receptor Tyrosine Kinase Down-regulation through Antibody-mediated Receptor Clustering

doi: 10.1038/s41598-018-36963-3

Figure Lengend Snippet: Antibody-mediated MET receptor crosslinking leads to the receptor clustering and patch formation on the plasma membrane through activation of MET receptor. ( A ) Live U373-MG cells, which were serum starved for three hours, were incubated with the APC-conjugated mouse monoclonal anti-MET antibody (1 μg/ml) (red) or its isotype control, APC-conjugated, mouse IgG (1 μg/ml) for 7 minutes. Cells were then washed, fixed and co-immunostained with an anti-MET antibody pre-conjuagted to Alexa Fluor 488, which detects the total MET protein (green). Cells were imaged by confocal microscopy. Clusters (or patches) of the MET proteins on the plasma membrane are indicated by arrows. ( B ) Live serum-starved U373-MG cells were treated with biotin-conjugated goat anti-MET antibodies (2 μg/ml) or biotin-conjugated normal goat IgG antibodies (2 μg/ml) (control) for 7 minutes. Cells were then washed, fixed and immunostained with the Streptavidin-conjugated to Brilliant Violet 421 (BV421) to detect the biotin-conjugated antibodies (purple), a rabbit anti-Y1234/1235-phosphorylated MET antibody for the activated MET protein (secondary antibody conjugated with Alexa Fluor 647, red), and an anti-MET antibody pre-conjugated to Alexa Fluor 488 for total MET protein (green). ( C ) Serum-starved cells were left untreated or stimulated with HGF (50 ng/ml) for 7 minutes. Cells were fixed and immunostained with a rabbit antibody against Y1234/Y1235-phosphorylated MET for the activated MET protein (red) and with an anti-MET antibody pre-conjugated with the Alexa Fluor 488 to detect total MET (green). ( D ) Serum-starved cells were treated with the biotin-conjugated anti-MET antibody (2 μg/ml) and fixed at the various time points as indicated. Cells were immunostained with strepavidin-conjugated to BV421 as in panel B. Scale bars in ( A–C ), 20 μm.

Article Snippet: Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT (at S473, recognizing all AKT isoforms, Cat. #9272), and anti-MET rabbit monoclonal antibody (clone D1C2, Alexa Fluor 488 conjugate, Cat. #8494) were obtained from Cell Signaling Technology.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Incubation, Control, Confocal Microscopy

Journal: Scientific Reports

Article Title: Induction of MET Receptor Tyrosine Kinase Down-regulation through Antibody-mediated Receptor Clustering

doi: 10.1038/s41598-018-36963-3

Figure Lengend Snippet: The MET receptor is activated by HGF, or APC- or Biotin-conjugated anti-MET antibodies. ( A ) Serum-starved U373-MG cells were treated either with HGF (50 ng/ml) for 7 minutes or left untreated (-HGF) as indicated. The cells were directly lysed in SDS buffer and proteins were detected by Western blotting with antibodies for the Y1234/Y1235-phosphorylated MET (p-MET, Y1234/5), total MET, phosphorylated AKT (p-AKT), total AKT, or actin (loading control), as indicated. ( B ) Serum-starved U373-MG cells were left untreated, or treated with APC-conjugated mouse IgG control antibody or APC-conjugated mouse monoclonal anti-MET antibody (each at 1 μg/ml) for 7 minutes. Cells were directly lysed in SDS buffer and proteins were detected by Western blotting with antibodies as in panel A. ( C ) The same as panel B except that the Biotin-conjugated goat control IgG or Biotin-conjugated goat anti-MET antibodies (each at 2 μg/ml) were used. ( D ) Serum-starved human glioblastoma cells T98G, LN229, and LN18 cells were treated with HGF or biotin-conjugated IgG or biotin-conjugated anti-MET antibodies as in panel A and C, or left untreated. The proteins were detected by Western blotting with antibodies as in panel A.

Article Snippet: Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT (at S473, recognizing all AKT isoforms, Cat. #9272), and anti-MET rabbit monoclonal antibody (clone D1C2, Alexa Fluor 488 conjugate, Cat. #8494) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Induction of MET Receptor Tyrosine Kinase Down-regulation through Antibody-mediated Receptor Clustering

doi: 10.1038/s41598-018-36963-3

Figure Lengend Snippet: Activation of the MET receptor by HGF or anti-MET antibodies requires the MET RTK activity. ( A ) Serum-starved U373-MG cells were treated with the MET kinase inhibitor, crizotinib (1 μM) (lane 4–6) or DMSO (lane 1–3) for 3 hours. The cells were then treated either with biotin-conjugated anti-MET antibodies or biotin-conjugated IgG (each at 2 μg/ml) for 7 minutes, or left untreated, as indicated. The cells were directly lysed in SDS buffer and proteins were detected by Western blotting with antibodies for the Y1234/Y1235-phosphorylated MET (p-MET, Y1234/5), total MET, phosphorylated AKT (p-AKT), total AKT, and actin (loading control), respectively, as in Fig. . ( B ) The same as in panel A except that the APC-conjugated mouse monoclonal anti-MET antibodies or mouse IgG control antibodies (each at 1 μg/ml) were used. ( C ) Serum-starved U373-MG cells were treated with dimethyl sulfoxide (DMSO) vehicle control (lane 1, 2), or with the MET kinase inhibitors crizotinib (1 μM) (lane 3, 4) or JNJ-38877605 (1 μM) (lane 5,6) for 3 hours. Cells were then treated with or without HGF (50 ng/ml) for 7 minutes as indicated. The cells were directly lysed in SDS buffer and proteins were detected by Western blotting with indicated antibodies. ( D ) Serum-starved U373-MG cells were treated with the MET kinase inhibitors, crizotinib, JNJ-38877605 or DMSO as in panel C. Cells were stimulated with or without HGF (50 ng/ml) for 7 minutes and were subsequently fixed and stained with rabbit antibody against Y1234/Y1235-phosphorylated MET (red) for the activated MET protein and with the Alexa Fluor 488 pre-conjugated anti-MET antibody for total MET protein (green), and bright field for the contrast phase. Cells were also counter stained with DAPI for nuclei. MET receptor clusters (patches) are indicated by arrows. ( E ) Serum-starved U373-MG cells were treated with the MET inhibitors crizotinib, JNJ-38877605 or dimethyl sulfoxide (DMSO) as in panel C. The live serum-starved U373-MG cells were then treated with biotin-conjugated goat anti-MET antibodies (2 μg/ml) or biotin-conjugated goat IgG antibodies (2 μg/ml) (control) for 7 minutes. The cells were then fixed and stained with streptavidin (labeled with Brilliant Violet 421) for biotin-conjugated antibodies (purple) and the rabbit anti-Y1234/1235-phosphorylated MET antibodies (followed by secondary antibodies conjugated to the Alexa Fluor 647) for the activated MET protein (red), and with the Alexa Fluor 488 pre-conjugated anti-MET antibody for total MET protein (green). MET receptor clusters (patches) are indicated by arrows. Scale bars in ( D,E ), 20 μm.

Article Snippet: Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT (at S473, recognizing all AKT isoforms, Cat. #9272), and anti-MET rabbit monoclonal antibody (clone D1C2, Alexa Fluor 488 conjugate, Cat. #8494) were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Western Blot, Control, Staining, Labeling

Journal: Molecular cancer therapeutics

Article Title: GnRH-R targeted lytic peptide sensitizes BRCA wild-type ovarian cancer to PARP inhibition

doi: 10.1158/1535-7163.MCT-18-0770

Figure Lengend Snippet: (A) A network of downregulated/upregulated proteins in combination group compared to olaparib group was determined using Netwalker analysis. (B) Expression of PI3K/AKT, p-PI3K/p-AKT, PARP, Cleaved PARP, BRCA1, BRCA2, and RAD 51 after treatment. (C) Expression and localization of GnRH-R in BRCA1 mutant (BRCA1m) COV362 and MDA-MB-436 cells and BRCA2 mutant (BRCA2m) KURAMOCHI cells. The fixed cells without permeabilization (left panels) were visualized using confocal microscopy (TCS SP5 MP; Leica Microsystems, Buffalo Grove, IL). The fixed and permeabilized cells (right panels) were visualized using a laser-scanning microscope (Leica). (Scale bar: 50 μM). (D) cell viability of COV362, MDA-MB-436, and KURAMOCHI cells after treatment with EP-100, olaparib, or EP-100 plus olaparib for 72 h. (E) CI values are shown as in Fa-CI plots.

Article Snippet: The primary antibody dilution factors were as follows according to manufacturer’s instructions: anti-GnRH-R (1:1,000, Abcam, Cambridge, UK, Cat: ab183079), RAD 51 (1:10,000, Abcam, Cambridge, UK, Cat: ab133534), phosphorylated PI3K p85 (1:1,000, Cat: 4228s), PI3K (1:1,000, Cat: 4292s), phosphorylated AKT Ser473 (1:1,000, Cat: 9271s), AKT (1:1,000, Cat: 9272s), PARP (1:1,000, Cat: 9532s), BRCA1 (1:1,000, Cat: 9025s) and BRCA2 (1:1,000, Cat: 10741s) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Mutagenesis, Confocal Microscopy, Laser-Scanning Microscopy

Journal: The Journal of Cell Biology

Article Title: Defective Rac-mediated proliferation and survival after targeted mutation of the β 1 integrin cytodomain

doi: 10.1083/jcb.200111065

Figure Lengend Snippet: Analysis by confocal microscopy of Erk nuclear accumulation. Cells were seeded on fibronectin in the presence of growth factors for 4 h with or without prior infection with Rac V12 or Rac N17 adenoviruses. Fixed and permeabilized cells were stained with an anti-Erk1/2 polyclonal antibody (C-14; Santa Cruz Biotechnology), bisbenzimide, and phalloidin-FITC. Bar, 10 μm.

Article Snippet: Cells were fixed with PFA 2%, permeated with TBS 0.3%-Triton, blocked with TBS 5%-BSA, stained ON at 4°C with a polyclonal antibody recognizing Erk (Cat: SC-16; Santa Cruz Biotechnology), washed again in TBS, and stained for 1 h with a rhodamine-conjugated anti–rabbit antibody mixed to a fluorescein-conjugated phalloidin.

Techniques: Confocal Microscopy, Infection, Staining

Journal: International journal of biological macromolecules

Article Title: Ubiquitin-conjugating enzyme E2S decreases the sensitivity of glioblastoma cells to temozolomide by upregulating PGAM1 via the interaction with OTUB2.

doi: 10.1016/j.ijbiomac.2025.140583

Figure Lengend Snippet: Fig. 2. UBE2S upregulates PGAM1 to decrease TMZ sensitivity in GBM cells. (A-D) The levels of protein UBE2S, PGAM1, γH2AX, caspase-3, and cleaved caspase-3 changes were examined by western blotting and analyzed after administration with TMZ (300 μM, 48 h) in U87 UBE2S-sh1 (A, B) and U251 UBE2S-sh1 (C, D) cell that transfected with plasmids expressing Myc-UBE2S or Flag-PGAM1 as indicated. (E-H) Apoptotic cells were detected by Flow cytometry after TMZ (300 μM, 48 h) treatment in U87 UBE2S-sh1 (E, F) and U251 UBE2S-sh1 (G, H) cells that transfected with plasmids expressing Myc-UBE2S or Flag-PGAM1 as indicated. Four in dependent experiments were performed. NS: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the cells were blocked for 1 h with 5 % Bovine Serum Albumin (BSA) and incubated overnight at 4 ◦C with primary antibodies against γ-H2AX (sc517,348, 1:250, Santa Cruz), UBE2S (sc-390,917, 1:300, Santa Cruz), or PGAM1 (Cat No. 16126–1-AP, 1:300, Proteintech).

Techniques: Western Blot, Transfection, Expressing, Flow Cytometry

Journal: International journal of biological macromolecules

Article Title: Ubiquitin-conjugating enzyme E2S decreases the sensitivity of glioblastoma cells to temozolomide by upregulating PGAM1 via the interaction with OTUB2.

doi: 10.1016/j.ijbiomac.2025.140583

Figure Lengend Snippet: Fig. 3. PGAM1 protein is positively regulated by UBE2S in glioma. (A, B) UBE2S and PGAM1 protein expression in normal brain and different grade glioma tissues from patients were examined by IHC. Scale bar, 50 μm. (C) UBE2S and PGAM1 protein expression were detected by western blotting in GBM tissues from four patients. N: non-cancerous tissues; T: tumor tissue. (D) Basic protein UBE2S and PGAM1 expression in normal astrocyte and different GBM cell lines were detected by western blotting. (E, G) PGAM1 protein level changed after UBE2S knockdown by two UBE2S-shRNAs (sh1/sh2) in U87 and U251 cells, as shown by western blotting. (F, H) PGAM1 protein levels changed as Flag-UBE2S re-expression in U87 UBE2S-knockdown (sh1) and U251 UBE2S-knockdown (sh1) cells seeded in 6-well plates, as examined by western blotting. Four independent experiments were performed. NS: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the cells were blocked for 1 h with 5 % Bovine Serum Albumin (BSA) and incubated overnight at 4 ◦C with primary antibodies against γ-H2AX (sc517,348, 1:250, Santa Cruz), UBE2S (sc-390,917, 1:300, Santa Cruz), or PGAM1 (Cat No. 16126–1-AP, 1:300, Proteintech).

Techniques: Expressing, Western Blot, Knockdown

Journal: International journal of biological macromolecules

Article Title: Ubiquitin-conjugating enzyme E2S decreases the sensitivity of glioblastoma cells to temozolomide by upregulating PGAM1 via the interaction with OTUB2.

doi: 10.1016/j.ijbiomac.2025.140583

Figure Lengend Snippet: Fig. 4. UBE2S interacts with PGAM1 and inhibits PGAM1 from proteasomal degradation. (A) Confocal microscopy showed protein UBE2S and PGAM1 distribution in U87 and U251 cells through IF staining. Scale bars, 10 μm. (B, C) The endogenous interactions between UBE2S and PGAM1 in U87 and U251 cells were detected by Co-IP and western blotting as immunoblotted with antibodies against the indicated proteins. (D, E) The exogenous interactions between UBE2S and PGAM1 in U87 and U251 cells were detected by Co-IP and western blotting after transfection with plasmids expressing Flag-PGAM1 or HA-UBE2S as indicated. (F, G) PGAM1 protein levels were measured by western blotting after incubation with or without 10 μM MG132 for 8 h in U87 (UBE2S-shNC, UBE2S-sh1) and U251 (UBE2S-shNC, UBE2S- sh1) cells. (H, I) CHX chase assays were performed to detect changes in PGAM1 protein levels after treatment with 50 μg/ml CHX in U87 (UBE2S-shNC, UBE2S-sh1) and U251 (UBE2S-shNC, UBE2S-sh1) cells, as examined by Western blotting. Four independent experiments were performed. NS: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the cells were blocked for 1 h with 5 % Bovine Serum Albumin (BSA) and incubated overnight at 4 ◦C with primary antibodies against γ-H2AX (sc517,348, 1:250, Santa Cruz), UBE2S (sc-390,917, 1:300, Santa Cruz), or PGAM1 (Cat No. 16126–1-AP, 1:300, Proteintech).

Techniques: Confocal Microscopy, Staining, Co-Immunoprecipitation Assay, Western Blot, Transfection, Expressing, Incubation

Journal: International journal of biological macromolecules

Article Title: Ubiquitin-conjugating enzyme E2S decreases the sensitivity of glioblastoma cells to temozolomide by upregulating PGAM1 via the interaction with OTUB2.

doi: 10.1016/j.ijbiomac.2025.140583

Figure Lengend Snippet: Fig. 5. UBE2S interacts with OTUB2 and decreases the polyubiquitination levels of PGAM1. (A) Co-IP and western blotting revealed changes in the ubiquitination levels of PGAM1 in 293T cells transfected with plasmids expressing HA-ub (WT, K11, K48, K63), Myc-UBE2S, or Flag-PGAM1 as indicated. (B–D) Co-IP and western blotting showed the interactions of PGAM1-OTUB2 (B), UBE2S-OTUB2 (C) and UBE2S-PGAM1-OTUB2 (D) in 293T cells that transfected with plasmids expressing Flag-PGAM1, HA-UBE2S, Myc-OTUB2 or Flag-UBE2S as indicated. (E) Co-IP and western blotting showed the interactions of PGAM1 and OTUB2 in 293T UBE2S-KO cells transfected with plasmids expressing Myc-OTUB2 or Flag-PGAM1 as indicated. (F) Co-IP and western blotting showed the K48-linked polyubiquitination of PGAM1 changes in 293T UBE2S-KO cells transfected with plasmids expressing Myc-UBE2S, Flag-PGAM1, Myc-OTUB2 or HA-ub-K48 as indicated.

Article Snippet: Subsequently, the cells were blocked for 1 h with 5 % Bovine Serum Albumin (BSA) and incubated overnight at 4 ◦C with primary antibodies against γ-H2AX (sc517,348, 1:250, Santa Cruz), UBE2S (sc-390,917, 1:300, Santa Cruz), or PGAM1 (Cat No. 16126–1-AP, 1:300, Proteintech).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Ubiquitin Proteomics, Transfection, Expressing

Journal: International journal of biological macromolecules

Article Title: Ubiquitin-conjugating enzyme E2S decreases the sensitivity of glioblastoma cells to temozolomide by upregulating PGAM1 via the interaction with OTUB2.

doi: 10.1016/j.ijbiomac.2025.140583

Figure Lengend Snippet: Fig. 6. UBE2S and OTUB2 are essential in regulating PGAM1 protein levels and its K48-linked deubiquitylation. (A) PGAM1 protein level changes after UBE2S or OTUB2 knockout in 293T cells as examined by western blotting. (B) PGAM1 protein level changes in 293T UBE2S-KO cells after transfecting with plasmids expressing HA-UBE2S or Myc-OTUB2 as indicated and examined by western blotting. (C) PGAM1 protein changes in OTUB2-KO 293T cells after transfecting with plasmids expressing HA-UBE2S or Myc-OTUB2 as indicated and examined by western blotting. (D) Co-IP and western blotting examined the level of K48-linked poly ubiquitination of PGAM1 changes in 293T UBE2S-KO cells transfected with plasmids expressing Myc-UBE2S, Flag-PGAM1, Myc-OTUB2 or HA-ub-K48 as indicated. (E) Co-IP and western blotting examined the level of K48-linked polyubiquitination of PGAM1 changes in 293T OTUB2-KO cells transfected with plasmids expressing Myc-UBE2S, Flag-PGAM1, Myc-OTUB2 or HA-ub-K48 as indicated. Four independent experiments were performed. NS: no significance, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the cells were blocked for 1 h with 5 % Bovine Serum Albumin (BSA) and incubated overnight at 4 ◦C with primary antibodies against γ-H2AX (sc517,348, 1:250, Santa Cruz), UBE2S (sc-390,917, 1:300, Santa Cruz), or PGAM1 (Cat No. 16126–1-AP, 1:300, Proteintech).

Techniques: Knock-Out, Western Blot, Expressing, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Transfection

Journal: International journal of biological macromolecules

Article Title: Ubiquitin-conjugating enzyme E2S decreases the sensitivity of glioblastoma cells to temozolomide by upregulating PGAM1 via the interaction with OTUB2.

doi: 10.1016/j.ijbiomac.2025.140583

Figure Lengend Snippet: Fig. 7. UBE2S knockdown improves TMZ efficacy in the orthotopic Gl261 allografts model in mice. (A) Schematic illustration of the Gl261 GBM cells orthotopic allograft model construction. (B) The cell viability was analyzed by the CCK-8 assay after UBE2S knockdown and treated with TMZ (300 μM) for 48 h in Gl261 cells. (C) Expression level changes of protein UBE2S, PGAM1, γH2AX, caspase-3, and cleaved caspase-3 after UBE2S knockdown and administered with TMZ (300 μM) for 48 h in Gl261 cells as determined by western blotting. (D) Tumors formation in mice brains of different groups were photographed 28 days after orthotopic im plantation, as indicated by dashed lines. (E) H&E staining showed the maximum cross section of tumor from different groups. Scale bar, 1 mm. (F) Tumor volume formation was calculated and analyzed. (G, H) UBE2S and PGAM1 expression were examined by IHC staining in different groups. Scale bar, 50 μm. (I, J) TUNEL staining, and analysis showed the apoptotic cells in different groups. The arrow indicated the positive apoptotic cells. Scale bar, 50 μm. Four independent experiments were performed. NS: no significance, * p < 0.05, ** p < 0.01, ***p < 0.001.

Article Snippet: Subsequently, the cells were blocked for 1 h with 5 % Bovine Serum Albumin (BSA) and incubated overnight at 4 ◦C with primary antibodies against γ-H2AX (sc517,348, 1:250, Santa Cruz), UBE2S (sc-390,917, 1:300, Santa Cruz), or PGAM1 (Cat No. 16126–1-AP, 1:300, Proteintech).

Techniques: Knockdown, CCK-8 Assay, Expressing, Western Blot, Staining, Immunohistochemistry, TUNEL Assay