Journal:

Article Title: Identification of functional domains on human interleukin 10

doi:

Figure Lengend Snippet: Predicted amino and carboxyl acid terminal sequences of murine IL-10, human IL-10, and viral IL-10 (2).

Article Snippet: Further, cells cultured with higher doses of IT9302, IT9403, or IL-10 were added 2 μg/ml of IL-10 sR monoclonal antibody (R & D Systems, MAB 274) 1 hr before adding IT9302, IT9403, or IL-10.

Techniques:

Journal:

Article Title: Identification of functional domains on human interleukin 10

doi:

Figure Lengend Snippet: The amino acid sequence of IT9403 residues 8–16 from hIL-10 and the homologous sequences from rat, murine, and viral IL-10.

Article Snippet: Further, cells cultured with higher doses of IT9302, IT9403, or IL-10 were added 2 μg/ml of IL-10 sR monoclonal antibody (R & D Systems, MAB 274) 1 hr before adding IT9302, IT9403, or IL-10.

Techniques: Sequencing

Journal:

Article Title: Identification of functional domains on human interleukin 10

doi:

Figure Lengend Snippet: IRAP production by IL-10- (A) or IT9302 (B)-stimulated human monocytes (24 hr). IRAP was measured by using an IRAP ELISA assay.

Article Snippet: Further, cells cultured with higher doses of IT9302, IT9403, or IL-10 were added 2 μg/ml of IL-10 sR monoclonal antibody (R & D Systems, MAB 274) 1 hr before adding IT9302, IT9403, or IL-10.

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Identification of functional domains on human interleukin 10

doi:

Figure Lengend Snippet: HLA-DR expression on human monocytes (flow cytometry) regulated by IFN-γ (10 ng/ml) alone or IL-10 (10 ng/ml) or IT9302 (1 ng/ml) added 1 hr before IFN-γ. In the same fraction the DNA content was measured as described in Materials and Methods, and the fraction of cells in apoptosis was calculated.

Article Snippet: Further, cells cultured with higher doses of IT9302, IT9403, or IL-10 were added 2 μg/ml of IL-10 sR monoclonal antibody (R & D Systems, MAB 274) 1 hr before adding IT9302, IT9403, or IL-10.

Techniques: Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy

doi: 10.1038/s41467-022-29401-6

Figure Lengend Snippet: a Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from 293 T cells co-transfected with indicated constructs. b , c IB analysis of WCL derived from 293 T cells co-transfected with indicated constructs. Cells were treated with 200 µg/ml cycloheximide (CHX) as indicated time points ( b ). PD-L1 band intensity was quantified by ImageJ, which was normalized to vinculin and then to the t = 0 time point ( c ). EV: empty vector. d – g IB analysis of WCL derived from sgControl or sg TRAF6 -treated H460 ( d ) or CT26 cells ( f ). PD-L1 mRNAs were analyzed using the RT-qPCR ( e , g ). h , i Representative images from IHC staining of PD-L1 and TRAF6 in human lung squamous carcinoma ( h ). Scale bar, upper panels: 300 μm; lower panels: 100 μm. Quantification of PD-L1 and TRAF6 staining intensities were performed by semi-quantitative scoring ( i ). n = 73, r = 0.3381, p = 0.0034; correlation coefficients were calculated using the Pearson test. Two-sided p -value was given. j IB analysis of WCL and anti-Flag IPs from 293T cells co-transfected with indicated Flag-TRAF constructs. k IB analysis of glutathione S-transferase (GST) pull-down precipitates from 293T cell lysates with ectopic expression of HA-PD-L1 incubated with bacterially purified recombinant GST or GST-TRAF6 protein. l A schematic illustration of TRAF6 protein sequence with different domains or truncated mutants. m IB analysis of WCL and anti-HA IPs from 293T cells co-transfected with indicated constructs. n IB analysis of WCL and Ni-NTA pull-down products derived from lysates of 293T cells co-transfected with indicated constructs. o IB analysis of WCL and Ni-NTA pull-down products derived from lysates of 293T cells co-transfected with indicated constructs. For j , m , n , and o , cells were treated with 10 µM MG132 for 12 h before harvesting. For c , e , and g data were presented as mean ± S.D. n = 3 biologically independent samples. Two-sided t -test. For a , j , k , and m – o , two independent experiments were conducted. The relevant raw data and uncropped dots are provided as a Source Data file.

Article Snippet: Anti-TRAF6 (H-274) rabbit mAb (sc-7221), Anti-USP8 (UBPY) (E-1) mouse mAb (sc-376130), anti-JNK (D-2) mouse mAb (sc-7345), and anti-RelA (5G8) mouse mAb (sc-81622) were purchased from Santa Cruz Biotechnology and diluted 1:1000.

Techniques: Western Blot, Derivative Assay, Transfection, Construct, Plasmid Preparation, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, Incubation, Purification, Recombinant, Sequencing

Journal: Cell stem cell

Article Title: TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity.

doi: 10.1016/j.stem.2021.12.007

Figure Lengend Snippet: Figure 6. MYC activation is regulated by TRAF6-dependent post-translational modifications (A) Differentially ubiquitinated substrates in TF-1 cells expressing a doxycycline (DOX) inducible shTRAF6 (+DOX) relative to control cells (DOX) (n = 3 inde- pendent experiments). (B) MYC ubiquitination was determined in HEK293 cells transfected with WT or K148R MYC and TRAF6. Shown is a representative image from three independent replicates. (C) MYC ubiquitination was determined in HEK293 cells transfected with WT or K63R ubiquitin (HA-Ub), MYC, and TRAF6. Percent of ubiquitinated MYC was determined using densitometric values by calculating ubiquitinated MYC relative to total immunoprecipitated MYC. Show is a representative image from three independent replicates. (D) Endogenous MYC ubiquitination was determined in Tet2/ and Traf6/;Tet2/ cKit+ BM cells. Percent of ubiquitinated MYC was calculated by determining the densitometric values of ubiquitinated MYC relative to total immunoprecipitated MYC. (E) MYC activation was measured by MYC-site containing reporter assays in HEK293 cells. Values are normalized to Renilla-luciferase and empty vector from the average of three independent experiments. Error bars represent the SEM. (F) mass spectrometry analysis of MYC post-translational modifications in AML. (G) Schematic of MYC oncogenic activation regulated by opposing TRAF6-dependent ubiquitination and acetylation at K148. (H) Immunoblot analysis of acetylated (K149) and phosphorylated (T58 and S62) MYC in c-Kit+ BM cells. Show is a representative image from three independent replicates.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-TRAF6 antibody (H-274) Santa Cruz Cat# sc-2003 Rabbit monoclonal anti-Vinculin antibody (E1E9V) Cell Signaling Technology Cat# 13901 Rabbit monoclonal anti-TET2 Antibody (D6B9Y) Cell Signaling Technology Cat# 18950 Mouse monoclonal anti- Ubiquitin antibody (P4D1) Santa Cruz Cat# sc-8017 Monoclonal anti- FLAG M2 antibody in mouse Sigma Cat# F1804 Rabbit polyclonal anti-c-Myc Antibody Cell Signaling Technology Cat# 9402 Rabbit monoclonal anti-phospho-c-Myc (Ser62) antibody (E1J4K) Cell Signaling Technology Cat# 13748 Rabbit monoclonal anti-phospho-c-Myc (Thr58/Ser62) antibody (E203) Millipore Cat# 04-217 Rabbit polyclonal anti-acetyl-c-Myc (Lys148) antibody Millipore Cat# ABE25 Rabbit monoclonal anti-GAPDH antibody (D16H11) Cell Signaling Technology Cat# 5174 Rabbit polyclonal anti-a/b-Tubulin antibody Cell Signaling Technology Cat# 2148 Rabbit monoclonal anti-Lamin B1 antibody (D4Q4Z) Cell Signaling Technology Cat# 12586 Normal Rabbit IgG Cell Signaling Technology Cat# 2729 Anti-CD11b PE-Cy7 eBioscience Cat# 25-0112-81 Anti-Gr1 eFluor450 eBioscience Cat# 48-5931-82 Anti-CD3 PE eBioscience Cat# 12-0031-83 Anti-B220 APC eBioscience Cat# 17-0452-82 Anti-Ter119 APC eBiosceince Cat# 17-5921-82 Anti-CD71 PE eBioscience Cat# 12-0711-81 Anti-CD45-APC-Cy7 BD Biosciences Cat# 557659 Anti-CD44-BV421 BD Biosciences Cat# 563970 Anti-CD45.1-Brilliant Violet 510 BioLegend Cat# 110741 Anti-CD45.2-APC-eFluor780 eBiosciences Cat# 47-0454-82, Anti-CD45.2- eFluor450 eBiosciences Cat# 48-0454-82 Mouse hematopoietic lineage biotin panel eBiosciences Cat# 88-7774-75 Anti-streptavidin eFluor780 eBiosciences Cat# 47-4317-82 Anti-Sca-1-PE eBiosciences Cat# 12-5981-82 Anti-c-Kit-APC eBiosciences Cat# 17-1171-81 Anti-CD48-FITC Affymetrix Cat# 11-0481-85 Anti-CD150-PE-cy7 BioLegend Cat# 115914 Anti-Sca-1-PE-cy7 eBiosciences Cat# 25-5981-82 Anti-CD34-FITC eBiosciences Cat# 11-0341-82 Anti-CD16/32-PE eBiosciences Cat# 12-0161-82 Anti-cMyc Cell Signaling Technology Cat # 18583S Bacterial and virus strains OneShot TOP10 Competent Cells ThermoFisher Cat# C404010 (Continued on next page) Cell Stem Cell 29, 298–314.e1–e9, February 3, 2022 e1

Techniques: Activation Assay, Expressing, Control, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Luciferase, Plasmid Preparation, Mass Spectrometry, Western Blot