Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) Expression of Rnf145 was evaluated in the indicated mouse tissues by qPCR. Bars indicate mean ± SD (n = 3) ( B , C ) The indicated ( B ) human and ( C ) mouse cell lines and primary macrophages were cultured in lipoprotein-depletion medium for 16 hours and subsequently treated for 6 hours with 1μM GW3965 (GW). ( D ) THP1 cells were cultured in sterol depletion medium for 16 hrs and then treated with 1μM GW3965 (GW) and 100nM LG100268 (LG) for 6 hrs as indicated. Subsequently, ABCA1 and RNF145 expression was determined by qPCR and each bar and error represents the mean fold-change of ligand-treated cells over sterol-depleted cells ± SD (n≥3).

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: Expressing, Cell Culture

Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) RAW264.7 macrophages were treated with 5μg/mL Actinomycin D (ActD) for the indicated time and expression of Abca1 and Rnf145 was determined by qPCR and plotted as mean ± SD relative to untreated cells (n = 3), ( B ) HepG2 and RAW264.7 cells were treated with 1μM GW3965 for 6 hours in the presence or absence of 5μg/ml actinomycinD for 4 hours, after which expression of the indicated genes was measured by qPCR. Bars indicate mean ± SD (n = 3) ( C , D ) THP1 macrophages were cultured in sterol-depletion medium for 16 hrs and then treated with ( C ) 1μM GW3965 (GW) for the indicated time, or ( D ) with the indicated concentration of GW3965 for 4 hrs. Subsequently, gene expression was evaluated qPCR and bars indicate mean ± SD (n = 3)

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: Expressing, Cell Culture, Concentration Assay, Gene Expression

Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) LXR ChIP-seq experiments in human THP1 cells (GSE28319) and RAW macrophage-like cells (GSE50944) were analyzed and used to identify active LXREs within the Rnf145/Rnf145 loci, as graphically illustrated. ( B , C ) Genomic location of the identified LXREs. In bold, nucleotides that were mutated to disrupt LXR binding ( C ) A 1kb genomic region upstream of the transcriptional start site of hRNF145 was cloned into a pGL3basic. The putative LXRE was also mutated as indicated above. The empty, RNF145 WT , RNF145 MUT , and ABCA1 reporter plasmids were co-transfected with or without RXRα and LXRα expression plasmids in HEK 293T cells. 24 hours post-transfection the cells were treated with 1μM GW3965 (LXR) and 100nM LG100268 (RXR) for 24 hours and measured for luciferase signal (n≥3). ( D ) Cells were transfected with an empty or a tandem LXRE-containing pGL2 as in C . In all luciferase experiments the transfection efficiency was normalized to co-transfected Renilla luciferase. Bars report normalized chemiluminescence relative to untreated control ± SD (n = 3).

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: ChIP-sequencing, Binding Assay, Clone Assay, Transfection, Expressing, Luciferase, Control

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) AML cell lines (KG1a and NB4 cells) were treated with DMSO (Ctrl), DAC (1 and 5 μM) or AZA (1 and 5 μM) for 48 hours. Soluble levels of MICA, MICB, ULBP1, ULBP2 and ULBP3 ligands were quantified by sandwich ELISA. Each bar represents the mean ± SEM of at least four independent experiments. * p < 0.05 and * p < 0.001. ( B ) KG1a and NB4 cells were treated with 5 μM of DAC for 48 hours. Before and after treatment, cell surface expression of NKG2DL was analyzed by flow cytometry using monoclonal antibodies specific to each NKG2DL (MICA, MICB, ULBP1, ULBP2 and ULBP3), followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and shaded grey histograms show the NKG2DL expression. Dotted vertical lines indicate mean fluorescence intensity value in untreated samples.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Bioprocessing, Incubation, Control, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) NKL cells were co-cultured with cell-free supernatants (sn) obtained from KG1a and NB4 cells untreated (sn-DMSO) or treated with 1 μM DAC for 48 hours (sn-DAC). NKL cells grow in culture medium were considered as a control (Ctrl). NKG2D expression was analyzed by flow cytometry and represented as mean fluorescence intensity (MFI). Each bar represents the mean ± SEM of three independent experiments. * versus control and p < 0.01; # versus sn-DMSO and p < 0.05. ( B ) NKL cells were co-cultured with K562 cells at the indicated E:T ratio in a cell lysis assay, in the absence (Ctrl) or presence of cellular supernatant derived from KG1a (left panel) and NB4 (middle panel) cells previously treated with DMSO (sn-DMSO) or 1 μM DAC (sn-DAC) for 48 hours. Specificity of the NKG2D-NKG2DL interaction was corroborated using an anti-NKG2D blocking mAb and the effect of DAC was assayed to analyze the non-specific effects on the lytic capacity of NKL cells (right panel). Measurements were made in duplicate and the mean ± SEM of the two independent experiments are shown. * versus control and p < 0.05; * versus control and p < 0.01; # versus sn-DMSO and p < 0.05.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Cell Culture, Control, Expressing, Flow Cytometry, Fluorescence, Lysis, Derivative Assay, Blocking Assay

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with inhibitors specific to ADAM17 (10 μM GW280264X) and ADAM10 (50 μM of GI254023X) for 48 hours. Levels of soluble NKG2DL (sMICA/B and sULBPs1-3) were quantified by sandwich ELISA. Values are the mean ± SEM of at least three independent experiments. * p < 0.05 and * p < 0.01. ( B ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (1 μM) for 48 hours. After treatment, cell surface expression of ADAM17 was analyzed by flow cytometry using anti-human ADAM17 monoclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and the shaded grey histograms show the ADAM17 expression. ( C ) ADAM17 activity in KG1a and NB4 cells was measured in whole-cell lysates after treatment with DMSO (Ctrl) or DAC (5 μM) for 48 hours. Data are expressed as relative fluorescence units (RLU) at Ex/Em=490/520 nm absorbance normalized with respect to micrograms of total protein (RLU/μg). Values are the mean ± SEM of three independent experiments. * p < 0.05 and * p < 0.01.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Incubation, Control, Activity Assay, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (0.25, 0.5 and 1 μM) for 48 hours, and TIMP3 expression was analyzed by qRT-PCR. Each bar represents the relative expression of TIMP3 normalized with respect to the reference gene (GAPDH), using the 2 −ΔCt method. MICA transcription levels in the KG1a cell line untreated (DMSO, Ctrl) or treated with DAC at different concentrations were used as a positive control. Results are summarized as the mean ± SEM of five independent experiments. * p < 0.05 and * p < 0.01. ( B ) TIMP3 protein levels were evaluated by western blot in KG1a and NB4 cells after treatment with DMSO (Ctrl) or DAC (1 μM or 5 μM) for 48 hours. * p < 0.05. ( C ) The TIMP3 methylation pattern was quantified by pyrosequencing in AML cell lines (KG1a and NB4 cells) before and after treatment with 1 μM or 5 μM DAC. Pie charts show the average percentage of methylation for the CpGs analyzed in the TIMP3 gene. ( D ) TIMP3 expression was inhibited by transfection of KG1a cells previously treated with DAC (1 μM) with a TIMP3-specific siRNA or nonspecific scramble siRNA (200 nM). * p < 0.05 ( E ) Soluble NKG2DL were quantified by sandwich ELISA after TIMP3 inhibition. Values shown are the mean ± SEM of three independent experiments. * versus control and p < 0.05; * versus control and p < 0.01 # versus nonspecific scramble siRNA and p < 0.05.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Methylation, Transfection, Sandwich ELISA, Inhibition, Control

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) Soluble NKG2DL were quantified by sandwich ELISA in sera from twelve AML patients before and after Vidaza ® treatment. Lines represent the levels of each sNKG2DL (ng/mL) before and after treatment of each individual AML patient. ( B ) Expression of NKG2DL on the cell surface of blasts from five AML patients before and after Vidaza ® treatment (left panel). The right panel shows dot plots of NKG2DL expression on the cell surface of blats from a representative patient. Numbers included in the figure quadrants indicate the percentage of positive cells for each NKG2DL.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 2. Characterization of the signaling capacity of Jagged1WT, Jagged1Ndr, Jagged1Slm and Jagged1Htu. (A)12XCSL-luciferease reporter activity in co- cultures of Notch-expressing cells (carrying 12XCSL-luc) and cells expressing wild-type or mutated Jagged1. Bar graphs show luciferase activity (arithmetic mean) from triplicates of each co-culture. Error bars indicate s.d.; ***P<0.001. (B)Western blot analysis of lysates from stable cell lines (Flp-in HEK293 cells) expressing no Jagged1 (–), Jagged1WT, Jagged1Ndr, Jagged1Slm, and Jagged1Htu show comparable levels of Jagged1 (reprobed with -actin as loading control). (C)Cell lines expressing Jagged1WT, but not Jagged1Ndr, Jagged1Slm and Jagged1Htu, bind N1ECD-Fc-Cy3 as assessed by FACS analysis of cells exposed to N1ECD-Fc-Cy3 for 1 hour. (D)Cells expressing Jagged1WT, but not Jagged1Ndr, Jagged1Slm and Jagged1Htu, bind N1ECD-Fc-Cy3 (red), whereas all four cell lines express Jagged1 (green) at the cell surface. Scale bar: 20m. (E)Streptavidin-precipitation of cell-surface biotinylated proteins (top panel; without biotin as control, bottom panel) from HEK293 cells stably expressing Jagged1WT, Jagged1Ndr, Jagged1Slm and Jagged1Htu. Reprobing for nicastrin shows that only the mature, cell-surface-bound form, is detected in the streptavidin-precipitated material (the five lanes to the right). (F)12XCSL-luciferease reporter activity from Notch-expressing cells (carrying 12XCSL-luc) co-cultured with cells expressing wild-type Jagged1 and increasing levels of Jagged1Ndr. No dominant-negative effect by Jagged1Ndr is observed.

Article Snippet: The following primary and secondary antibodies were used in the study: rabbit antiJagged1 (Santa Cruz), goat anti-Jagged1 (Santa Cruz), rabbit anti-pan Cadherin (BD Biosciences), mouse anti-HA (Nordic Biosite and Covance), rabbit anti-GFP (Abcam), rat anti-PECAM (BD Biosciences), rabbit anti-nicastrin (Sigma), anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen) anti-mouse Cy3 (Jackson), anti-mouse Cy5 (Jackson), anti-rabbit Alexa Fluor 488 (Molecular Probes/Invitrogen), anti-goat Alexa Fluor 488 (Molecular probes/Invitrogen), anti-goat Cy3 (Jackson) anti-human Cy3 (Jackson), anti-human Cy5 (Jackson), anti-rat HRP (Vector Laboratories), antigoat HRP (DAKO), anti-mouse HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques: Activity Assay, Expressing, Luciferase, Co-Culture Assay, Western Blot, Stable Transfection, Control, Cell Culture, Dominant Negative Mutation

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 3. Mib1 and endocytosis of Jagged1 are required for activation of, but not binding to, Notch receptors. (A,B)12XCSL-luciferease reporter activity in full-length Notch1-expressing cells co-cultured with cells transiently transfected with (A) EGFP, Jagged1+EGFP or Jagged1+EGFP-tagged DynaminII K44A as indicated or (B) control siRNA, Jagged1+control siRNA or Jagged1+siRNA specific for MIB1 as indicated. Bar graphs show luciferase activity (arithmetic mean) from triplicates of each co-culture. Luciferase activity was normalized to control cultures transfected with EGFP (A) or control siRNA (B). Error bars indicate s.d.; **P<0.01. (C)Immunocytochemistry demonstrating that cells transfected with DynaminII K44A and Jagged1 (top cell) can bind N1ECD-Fc-Cy3, whereas cells not transfected with DynaminII K44A and Jagged1 (the bottom cell) does not bind N1ECD-Fc-Cy3. As a control for block of endocytosis by DynaminII K44A, there is no transferrin uptake in the top (DynaminII K44A expressing) cell, whereas transferrin is internalized in the bottom cell. Scale bar: 10m. Immunofluorescence images were adjusted using the levels function in Photoshop. In each case, adjustments were applied to the entire image. (D)Detection of ligand-receptor interaction by flow cytometry in cells transiently transfected with empty plasmid and EGFP, Jagged1 and EGFP or DynaminII K44A and EGFP, as indicated. (E)Detection of ligand-receptor interaction by flow cytometry in cells transiently transfected with empty plasmid and control siRNA, Jagged1 and control siRNA or Jagged1 and MIB1 siRNA, as indicated. Percentages of fluorescently labeled cells (arithmetic mean) is shown (bar graphs). Error bars indicate s.d.; *P<0.05, **P<0.01.

Article Snippet: The following primary and secondary antibodies were used in the study: rabbit antiJagged1 (Santa Cruz), goat anti-Jagged1 (Santa Cruz), rabbit anti-pan Cadherin (BD Biosciences), mouse anti-HA (Nordic Biosite and Covance), rabbit anti-GFP (Abcam), rat anti-PECAM (BD Biosciences), rabbit anti-nicastrin (Sigma), anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen) anti-mouse Cy3 (Jackson), anti-mouse Cy5 (Jackson), anti-rabbit Alexa Fluor 488 (Molecular Probes/Invitrogen), anti-goat Alexa Fluor 488 (Molecular probes/Invitrogen), anti-goat Cy3 (Jackson) anti-human Cy3 (Jackson), anti-human Cy5 (Jackson), anti-rat HRP (Vector Laboratories), antigoat HRP (DAKO), anti-mouse HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques: Activation Assay, Binding Assay, Activity Assay, Expressing, Cell Culture, Transfection, Control, Luciferase, Co-Culture Assay, Immunocytochemistry, Blocking Assay, Immunofluorescence, Flow Cytometry, Plasmid Preparation, Labeling

Journal: Journal of cell science

Article Title: Control of Notch-ligand endocytosis by ligand-receptor interaction.

doi: 10.1242/jcs.073239

Figure Lengend Snippet: Fig. 7. NECD is transendocytosed into Jagged1WT-expressing cells and transported through the endocytic degradation pathway. (A)HEK293T cells expressing full length HA-Notch1 (N-terminally tagged Notch1) were co-cultured with Jagged1WT-expressing HEK293T cells transfected with EGFP-EEA1. HA immunoreactivity was observed in EGFP-positive and Jagged1-positive vesicles indicating trans-endocytosis of Notch1 ECD into EEA1-positive vesicles in Jagged1-expressing cells. Boxed region is magnified in inset. Scale bar: 20m. (B)Jagged1WT cells exhibit high levels of trans-endocytosis of Notch1 ECD, whereas Jagged1Ndr cells exhibit low-to-no trans-endocytosis of Notch1ECD into Jagged1- and EEA1-positive vesicles (arrowheads). Scale bar: 20m. (C-E)Immunocytochemistry of cells with fluorescently labeled subcellular markers and incubated with N1ECD-Fc-Cy3. (C)Immediately after incubation, N1ECD-Fc-Cy3 (red) colocalized with the plasma-membrane marker EGFP-F. (D)At 1 hour, N1ECD-Fc-Cy3 (red) was detected in EGFP-EEA1-positive vesicles. (E)At 4 hours, N1ECD-Fc-Cy5 (green) was detected in lysosomes, as visualized by the lysosome marker Lysotracker Red (red).

Article Snippet: The following primary and secondary antibodies were used in the study: rabbit antiJagged1 (Santa Cruz), goat anti-Jagged1 (Santa Cruz), rabbit anti-pan Cadherin (BD Biosciences), mouse anti-HA (Nordic Biosite and Covance), rabbit anti-GFP (Abcam), rat anti-PECAM (BD Biosciences), rabbit anti-nicastrin (Sigma), anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen) anti-mouse Cy3 (Jackson), anti-mouse Cy5 (Jackson), anti-rabbit Alexa Fluor 488 (Molecular Probes/Invitrogen), anti-goat Alexa Fluor 488 (Molecular probes/Invitrogen), anti-goat Cy3 (Jackson) anti-human Cy3 (Jackson), anti-human Cy5 (Jackson), anti-rat HRP (Vector Laboratories), antigoat HRP (DAKO), anti-mouse HRP (DAKO), and anti-rabbit HRP (DAKO).

Techniques: Expressing, Cell Culture, Transfection, Immunocytochemistry, Labeling, Incubation, Clinical Proteomics, Membrane, Marker

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the expression, cellular presentation, function and analysis of the soluble human hetIL-15 complex. a. Engineered human IL-15 and IL-15Rα were co-expressed and secreted by HEK293 cells as a soluble heterodimeric complex (hetIL-15) after proteolytic cleavage from the cell surface. The complex binds to the IL-2Rβ/γ receptor complex located on target cells, where it initiates a cellular response. b. The hetIL-15 complex was isolated and its purity monitored using reducing (upper) and native (lower) SDS-PAGE with Coomassie blue staining (left gels) and Western blotting using anti-IL-15 and anti-IL-15Rα antibodies (right gels). The two clinically relevant preparations of hetIL-15 (i.e. the EN and cGMP lots) are shown. c. IL-15 and sIL-15Rα were separated from their heterodimeric complex by non-reductive RP-HPLC and individually subjected to LC-MS/MS-based glycan (top), glycopeptide (bottom) and glycoprotein (right) profiling in order to characterize their N- and O-glycosylation in a detailed and site-specific manner

Article Snippet: Slightly different conditions were used to separate the two examined hetIL-15 preparations ( i.e. , EN and cGMP lots): The EN lot of hetIL-15 was separated at a flow rate of 5 ml/min on a preparative PLRP-S polymeric C18 RP-HPLC column (i.d., 50 mm, length, 150 mm, Agilent).

Techniques: Expressing, Isolation, SDS Page, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: An intermediate state in trans-differentiation with proliferation, metabolic, and epigenetic switching

doi: 10.1016/j.isci.2021.103057

Figure Lengend Snippet:

Article Snippet: Primary antibody of rabbit monoclonal anti-Col1a1 (bs-20124R) (1:500 dilution) was purchased from Bioss Antibodies.

Techniques: Recombinant, Isolation, Transfection, Sequencing, Software