histidine tagged fusion proteins  (R&D Systems)

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification and characterization of an alternative cancer-derived PD-L1 splice variant

doi: 10.1007/s00262-018-2284-z

Figure Lengend Snippet: a) Genomic track showing a previously published case whereby HPV16 was found to be integrated in the intron between exon 4 and exon 5 of the PD-L1 locus, resulting in expression of a b) transcript with an alternative 3’ region including a readthrough of 56 nucleotides into the intron after exon 4, including a stop codon and an alternative polyadenylation signal. The predicted difference in amino acid sequences is depicted (b, c), with an alternative 18 amino acid sequence after exon 4 [1]

Article Snippet: Standard curves were made with human PD-L1-histidine tagged fusion protein (Sino Biological, # 10084-H08H).

Techniques: Expressing, Sequencing

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification and characterization of an alternative cancer-derived PD-L1 splice variant

doi: 10.1007/s00262-018-2284-z

Figure Lengend Snippet: a) The expression of PD-L1 isoforms with high exon 4/5 expression ratios was measured in 33 different cancer types in the TCGA dataset and corresponding normal tissue from GTEx. The x-axis represents the log2 expression of total PD-L1. The y-axis represents the PD-L1 exon 4/5 expression ratio, which is the expression of the fourth exon of PD-L1 over the expression of the fifth exon of PD-L1. The HNSCC index case is labeled (TCGA_CV_5443). b) Violin plot of expression of PD-L1 isoforms with high exon 4/5 expression ratios in cancer types ordered by decreasing median PD-L1 expression. Observed differences are significant as determined by an ANOVA comparison of the means (p-value < 0.0001). Cancer type abbreviations are defined in the materials and methods section. c) Cancer cell lines were analyzed for the expression of exon 4-enriched PD-L1 as described in a), from the CCLE dataset. The y-axis represents the log2 expression. The top two cell lines (RKO, blue; CAL62, green) with the highest PD-L1 exon 4/5 expression ratio as well as with the highest total PD-L1 expression were chosen for protein verification. RERF-LC-Ad1 (red, arrow) was chosen as a negative control, given it had high PD-L1 expression and a low PD-L1 exon 4/5 expression ratio. d, e) Media from RKO, RERF-LC-Ad1, CAL62, and HEK293T (negative control) was analyzed for PD-L1 protein expression. d) Media was used in an ELISA to measure PD-L1 present in the media. Data is from two separate experiments. The mean and SEM are plotted. e) Protein was precipitated from media with TCA precipitation or isolated in cell lysates and analyzed with western blotting for PD-L1 protein expression. Vinculin was used as a loading control.

Article Snippet: Standard curves were made with human PD-L1-histidine tagged fusion protein (Sino Biological, # 10084-H08H).

Techniques: Expressing, Labeling, Negative Control, Enzyme-linked Immunosorbent Assay, TCA Precipitation, Isolation, Western Blot

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification and characterization of an alternative cancer-derived PD-L1 splice variant

doi: 10.1007/s00262-018-2284-z

Figure Lengend Snippet: a) Diagram depicting the differential sequences of sec-PD-L1-long and sec-PD-L1-short. b, c) PD-L1-HA, sec-PD-L1-long-HA, sec-PD-L1-short-HA were expressed in HEK293T cells and PD-L1 protein in media was measured by b) ELISA. Data is from three separate experiments. c) PD-L1 was measured in media using TCA precipitation and in cell lysates by western blot by probing for HA tag. Vinculin was used as a loading control. Densitometry was performed where the HA signal was normalized to vinculin and the ratio of HA signal in the media versus lysate is depicted. d, e) HEK293T cells were transfected with empty vector (pcDNA3.1(1)), PD-L1-HA, sec-PD-L1-long-HA and sec-PD-L1-short-HA, and 2 days later were d) stained for PD-L1 (PE), and surface PD-L1 was analyzed with flow cytometry. Duplicates were performed, and 10,000 events were collected. The percent PD-L1 positive cells are shown. Data is representative of multiple experiments. e) Cells were immunofluorescently stained for HA tag (green), β-catenin (red), and with Hoescht (blue), and imaged with a fluorescent microscope. 10 um scale bar is shown

Article Snippet: Standard curves were made with human PD-L1-histidine tagged fusion protein (Sino Biological, # 10084-H08H).

Techniques: Enzyme-linked Immunosorbent Assay, TCA Precipitation, Western Blot, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Microscopy

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification and characterization of an alternative cancer-derived PD-L1 splice variant

doi: 10.1007/s00262-018-2284-z

Figure Lengend Snippet: a) PD-1 binding capacity of IgG1-FC (negative control), rhPD-L1-FC (positive control) or sec-PD-L1-long-FC was measured using a functional ELISA, where mean OD was measured at varying concentrations of IgG1-FC, rhPD-L1-FC or sec-PD-L1-long-FC protein. Kd values are displayed. The corresponding Scatchard graph displaying the differences in slope (−1/Kd) of rhPD-L1-FC (medium gray) or sec-PD-L1-long-FC (light gray) is shown as an inset. Data is from two separate experiments. b) The ability of PD-L1 and PD-1 neutralizing antibodies (10ug/ml) to block PD-1 binding to rhPD-L1-FC and sec-PD-L1-long-FC was tested with a functional ELISA. An IgG1 isotype control antibody was used as a negative control. Normalized mean OD values compared to incubation with the appropriate IgG1 isotype control conditions are plotted. Data is from two separate experiments. A two-way ANOVA was used. The mean and SEM are plotted. c, d) Primary human T cell blasts were incubated with IgG1-FC (negative control), rhPD-L1-FC (positive control) or sec-PD-L1-long-FC at either 20ug/ml or 40ug/ml in the presence of anti-CD3 for 24 hours, and media was harvested to quantify c) IL-2 and d) IFNg with ELISA. Samples were run in duplicate. N=5. Normalized protein levels compared to the appropriate IgG1-Fc control are plotted. A one-way ANOVA was used. The mean and SEM are plotted. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: Standard curves were made with human PD-L1-histidine tagged fusion protein (Sino Biological, # 10084-H08H).

Techniques: Binding Assay, Negative Control, Positive Control, Functional Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation