Journal: Cancers

Article Title: LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma

doi: 10.3390/cancers12082080

Figure Lengend Snippet: LncRNA DLG2-AS1 is downregulated in lung adenocarcinoma (LUAD). ( a ) heatmap of the 90 lncRNAs analyzed with the Human LncRNA Profile Kit from System Biosciences (SBI); ( b ) DLG2-AS1 expression in normal and tumor samples; and ( c ) tumor/normal fold change (FC(T/N)) of DLG2-AS1 expression in the 65 LUAD patients. Shown in a darker color are the patients who presented DLG2-AS1 downregulation (FC(T/N) < 0.66). *** = p -value < 0.001.

Article Snippet: Human DLG2 protein was detected using the anti-PSD-93 antibody (CAT# APZ-002, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing

Journal: Cancers

Article Title: LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma

doi: 10.3390/cancers12082080

Figure Lengend Snippet: DLG2-AS1 restoration model on lung adenocarcinoma (LUAD) cell lines successfully overexpresses DLG2-AS1. DLG2-AS1 relative expression was measured by RT-qPCR 5 days after transfection, and normalized by the expression of the reference gene U1 snRNA. In all three cell lines tested, DLG2-AS1 was successfully overexpressed (relative expression (pLVX-DLG2AS/EV) > 2). * = p -value < 0.05.

Article Snippet: Human DLG2 protein was detected using the anti-PSD-93 antibody (CAT# APZ-002, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Cancers

Article Title: LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma

doi: 10.3390/cancers12082080

Figure Lengend Snippet: DLG2-AS1 is not a cis-regulator of DLG2 expression. ( a ) RNA expression levels of DLG2 were unaffected after DLG2-AS1 overexpression (pLVX-DLG2AS1) compared to empty vector transfection (EV). Results from RT-qPCR were normalized to expression of the reference gene U1 snRNA. ( b ) Protein levels of human DLG2 did not change upon transfection with pLVX-DLG2AS1 compared to EV in two independent Western blot experiments (relative pLVX-DLG2AS1/EV densitometry measure mean = 0.984 ± 0.199, p = 0.929). Uncropped WB images are available at the .

Article Snippet: Human DLG2 protein was detected using the anti-PSD-93 antibody (CAT# APZ-002, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, RNA Expression, Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma

doi: 10.3390/cancers12082080

Figure Lengend Snippet: DLG2-AS1 shows potential as a lung adenocarcinoma (LUAD) biomarker: ( a ) receiver operating characteristic (ROC) curve and area under curve (AUC) value of DLG2-AS1 expression in our cohort of LUAD patients; and ( b ) ROC curves and AUC values of other LUAD biomarkers (EGFR, TP53, MALAT-1, and NEAT1), obtained from TCGA data.

Article Snippet: Human DLG2 protein was detected using the anti-PSD-93 antibody (CAT# APZ-002, Alomone Labs, Jerusalem, Israel).

Techniques: Biomarker Assay, Expressing

Journal: Cancers

Article Title: LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma

doi: 10.3390/cancers12082080

Figure Lengend Snippet: Sequences of used oligonucleotides for qPCR and vector cloning.

Article Snippet: Human DLG2 protein was detected using the anti-PSD-93 antibody (CAT# APZ-002, Alomone Labs, Jerusalem, Israel).

Techniques: Plasmid Preparation, Clone Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: ProBDNF stably expressed in HEK293F cells is glycosylated. N-Glycosylation of proBDNF and the prodomain is documented by mass shift following deglycosylation with PNGase F: A, detection of proBDNF and BDNF by an antibody recognizing the BDNF region; B, detection of proBDNF and the prodomain by an antibody recognizing the prodomain region. The gel images were spliced as indicated by space to exclude samples not related to the study.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Extracted ion chromatograms (XIC) of (A) nonglycosylated, NYLDAANMSMR and (B) deglycosylated, NYLDAADMSMR glycopeptide of proBDNF secreted by HEK293F cells confirm site occupancy higher than 99%. C, tandem mass spectrum verifies deglycosylation of the proBDNF peptide under [18O]water.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Expression of wildtype (WT) proBDNF and its N121Q mutant (NQ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media (A) and cell lysates (B). Arrows point to (pro)BDNF forms: glycosylated proBDNF (black arrow), nonglycosylated proBDNF (white arrow), and mature BDNF (gray arrow). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR (C). ProBDNF/BDNF expression in cell lysates (D) or conditioned media (E) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 (MG), lysosomal inhibitor chloroquine (CQ), or chemical chaperone 4-phenylbutyric acid (PBA). The black arrow points to the position of nonglycosylated proBDNF.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Expressing, Mutagenesis, Stable Transfection, Transfection, Western Blot, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Kinetics of cleavage of glycosylated and nonglycosylated proBDNF by furin. Mass difference between glycosylated and nonglycosylated proBDNF enabled densitometric quantification of each proBDNF form: A, representative blot of the cleavage kinetic at the indicated time intervals; B, densitometric analysis of proBDNF intensities at the indicated time intervals. Results are expressed as percent of the proBDNF concentration (mean ± S.D., n = 3) at the beginning of the reaction. Note that proBDNF without C-terminal Myc-FLAG tag was used in the reaction as described under “Experimental procedures.”

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Concentration Assay, FLAG-tag

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Mass spectra of proBDNF under the following conditions. A, precursor profile of the tryptic glycopeptide NYLDAANM(ox)SM(ox)R of proBDNF expressed in HEK293F cells; B, HCD fragmentation spectrum of the major glycoform (m/z 1132.42) at low collision energy (10% NCE) showing that characteristic LacdiNAc oxonium ion HexNAc-HexNAc (m/z 407.17) and its fucosylated form (m/z 553.22) with complementary y-ions (m/z 1373, 1381, 1454, and 1555) dominate the composition.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Relative abundance of the glycoforms of HEK293F-produced proBDNF and prodomain expressed as % of all identified glycoforms

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Produced

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Extracted ion chromatograms of four of the most abundant glycopeptides of proBDNF expressed in PC12 cells. A, C, and D, LacdiNAc containing structures; B, LacNAc containing structure. The arrow points to the peaks of glycopeptides aligned with slight retention time shifts as expected based on the hydrophilicity of the attached glycan. Numbers represent intensity of the extracted peaks (×105).

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Impact of modulators of glycosylation on the BDNF/proBDNF ratio in HEK293F cells. Western blotting using antibodies to prodomain and mature BDNF regions was analyzed by densitometry for the following: A, intracellular BDNF to proBDNF ratio; B, secreted BDNF to proBDNF ratio; C, intracellular prodomain to proBDNF ratio; and D, secreted prodomain to proBDNF ratio. The representative Western blotting is labeled as the graphs: CTRL, control; CHLOR, 50 mm sodium chlorate; KIF, 1 μg/ml kifunensine; TNMC, 5 μg/ml of tunicamycin. Results are expressed as scatter plot (mean ± S.D., n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with CTRL, one-way analysis of variance with Bonferroni adjustment.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Western Blot, Labeling