Journal: Scientific Reports

Article Title: Combining DNMT and HDAC6 inhibitors increases anti-tumor immune signaling and decreases tumor burden in ovarian cancer

doi: 10.1038/s41598-020-60409-4

Figure Lengend Snippet: DNMT1 protein levels are decreased by combination treatment of DNMTi and HDAC6i. ( A ) Ovarian cancer cell lines were treated as in Fig. and protein was extracted at Day 7 after treatment with IFN-gamma (IFN-γ+) (to assess MHC I and PD-L1 expression, in later figures) or control (IFN-γ -). Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( B ) The TykNu cell line was treated as in ( A ) and the protein synthesis cycloheximide added to cells on Day 7 for 0, 4, and 8 hours at 10 μM as indicated on the blot. Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( C ) Stable knockdowns of the HDAC6 protein were generated in the ID8 Trp53+/+ and Trp53−/− cell lines . Protein was extracted and immunoblots were run for the DNMT1 protein with B-actin as a loading control. Immunoblot membranes were probed for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( D ) Immunoblot showing knockdown of HDAC6 protein with a-Tubulin as a loading control. Protein was extracted and immunoblots were run for the HDAC6 protein with B-actin as a loading control. Immunoblots were probed for HDAC6 (131 kDa) and tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( E ) Ovarian cancer cell lines were treated as in Fig. and RNA was extracted at Day 7. qRT-PCR was run for DNMT1, DNMT3a, and DNMT3b and TBP was used as a reference gene. *p < 0.05 compared to Mock.

Article Snippet: The antibodies used for immunoblotting included: DNMT1 (Sigma, D4692), PD-L1 (ProSci, 4059), HDAC1 (Cell Signaling, 2062), HDAC2 (Cell Signaling, 2540), HDAC6 (Assay Biotech, C0026), acetyl-alpha Tubulin (Cell Signaling, 3971), alpha-Tubulin (Cell Signaling, 3873).

Techniques: Expressing, Control, Isolation, Western Blot, Generated, Knockdown, Quantitative RT-PCR

Journal: Immunity

Article Title: Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells

doi: 10.1016/j.immuni.2021.03.007

Figure Lengend Snippet:

Article Snippet: VioBright 515 anti-human PD-1 (CD279) , Miltenyi , AB_2752077.

Techniques: Purification, Control

Journal: Cellular and Molecular Immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Computer-aided homology modeling. (a) The ribbon structure of the human PD-1 and PD-L1 complex based on the computer-guided modeling and docking methods. (b) The fine structure of the binding mode between human PD-1 and PD-L1. The purple sticks denote the key amino-acid residues in human PD-L1, whereas the green balls and sticks denote the key amino-acid residues in human PD-1. (c) The 3-D theoretical structure of IgHV3-33*01, where the balls and sticks denote H28, H31 and H32. (d) The 3-D theoretical structure of IgHK1-11*01, where the balls and sticks denote L27, L50 and L91.

Article Snippet: The CD274 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014143","term_id":"1519243726","term_text":"NM_014143"}} NM_014143 ) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Binding Assay

Journal: Cellular and Molecular Immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Preliminary functional assessment of FV78. (a) Binding activity of FV78 to PD-1/Fc. (b) Epitope comparability of FV78 and MIL75 binding to PD-1/Fc by ELISA assay. (c) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L1 to PD-1 by ELISA assay. (d) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L1 to PD-1 by FACS assay. (e) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L2 to PD-1 by ELISA assay. (f) Evaluation of the cross-reactive binding activity of FV78 to other CD28 family members.

Article Snippet: The CD274 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014143","term_id":"1519243726","term_text":"NM_014143"}} NM_014143 ) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Functional Assay, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay