Journal: Cell reports
Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.
doi: 10.1016/j.celrep.2020.108073
Figure Lengend Snippet: Figure 3. BIRC2-KD in Melanoma Cells Upre- gulates CXCL9 Via Non-canonical NF-kB Pathway (A) B16F10 subclones expressing NTC or one of five shRNAs targeting BIRC2 were cultured at 20% O2 and analyzed for expression of BIRC2 protein by immunoblot assay. (B) Volcano plot showing expression of chemokines and cytokines in BIRC2-KD subclone compared with NTC (color coding of results is shown at the right). (C and D) Analysis of CXCL9 mRNA expression by qRT-PCR (C) and protein expression by ELISA (D) in B16F10 subclones, which expressed NTC or BIRC2 shRNA (sh3 or sh4). Data were normalized to NTC (mean ± SEM; n = 3); *p < 0.01 versus NTC (one- way ANOVA with Bonferroni post-test). (E) Immunoblot assays for RelA, RelB, p50, and a-tubulin were performed on cytosolic and nuclear fractions of cell lysates, which were prepared from B16F10 subclones. a-Tubulin is a cytosolic protein that was analyzed to demonstrate the purity of the nuclear lysates. See also Table S2.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020
Techniques: Expressing, Cell Culture, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, shRNA
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