Recombinant Human C20orf196, GST-tagged

• Cat.No.: C20orf196-10497H
• Product Overview: Recombinant Human C20orf196 protein, fused to GST-tag, was expressed in E.coli and purified by GSH-sepharose.

Specification

• Species: Human
• Source: E.coli
• Tag: GST
• Protein Length: 1-141a.a.
• Storage: The protein is stored in PBS buffer at -20℃. Avoid repeated freezing and thawing cycles.
• Storage Buffer: 1M PBS (58mM Na2HPO4,17mM NaH2PO4, 68mM NaCl, pH8. ) added with 100mM GSH and 1% Triton X-100,15%glycerol.

Gene Information

• Gene Name: C20orf196 chromosome 20 open reading frame 196 [ Homo sapiens ]
• Official Symbol: C20orf196
• Synonyms: C20ORF196; chromosome 20 open reading frame 196; uncharacterized protein C20orf196; FLJ25067; RP4-784N16.1
• Gene ID: 149840
• mRNA Refseq: NM_152504
• Protein Refseq: NP_689717
• UniProt ID: Q8IYI0
• Chromosome Location: 20p12.3

Citation

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells

Journal: Nature cell biology    PubMed ID: 30022119    Data: 2018/9/14

Authors: Harveer Dev, Ting-Wei Will Chiang, Stephen P. Jackson

Article Snippet:Gluathione sepharose beads (GE Healthcare) were washed with ice-cold PBS and blocked for 30min with PBS supplemented with 10% bacterial lysate (non-induced BL21 cells, lysed using PBS/lysozyme) then resuspended in binding buffer (10mM Tris pH7.5, 150 mM NaCl, 0.5% NP40, 0.5 mM EDTA, 0.5 % BSA).Gluathione sepharose beads (GE Healthcare) were washed with ice-cold PBS and blocked for 30min with PBS supplemented with 10% bacterial lysate (non-induced BL21 cells, lysed using PBS/lysozyme) then resuspended in binding buffer (10mM Tris pH7.5, 150 mM NaCl, 0.5% NP40, 0.5 mM EDTA, 0.5 % BSA).. Purified GST (bacterial expression), GST-FAM35A (Novus Biologicals), and His-C20orf196 (Creative BioMart) were added to the beads at 2 pmol and incubated for 30min at 4°C.. Beads were washed 5x with 10 mM Tris, pH 7.5, 250 mM NaCl, 0.5 % NP40, 0.5 mM EDTA and eluted with 100 mM Tris pH 8, 20 mM reduced glutathione, 120 mM NaCl for 15min rotating at 4°C.Beads were washed 5x with 10 mM Tris, pH 7.5, 250 mM NaCl, 0.5 % NP40, 0.5 mM EDTA and eluted with 100 mM Tris pH 8, 20 mM reduced glutathione, 120 mM NaCl for 15min rotating at 4°C.

a, Schematic of screen procedure. b, MAGeCK analysis of guide enrichments following specified drug treatments; false discovery rate (FDR) of 0.1 indicated by dotted line; n=3 technical replicates per drug treatment. c, siRNA mediated verification of hits in clonogenic survival assays; lower panels show area under the curve (AUC); n=3 independent experiments d, De novo Cas9 mediated knockout (ko) verification and complementation for FAM35A in clonogenic survival assays (multiple ko clones are shown in AUC); n=4 independent experiments except FAM35Ako(#14) (n=2), FAM35Ako(#40) (n=3), BRCA1ko/FAM35Ako(#34) (n=2), and BRCA1ko/FAM35Ako(#2) +FAM35A (n=3). e, As (d) but for C20orf196; n=3 independent experiments except BRCA1ko/C20orf196ko + C20orf196 (n=2). c-e Bars represent mean ± SEM, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05). Individual data points are plotted over bars, and statistical source data including the precise p values can be found in .

a, FAM35A and C20orf196 predicted domains and variants used, OB fold (OB), FAM domain (OB3/FD). b, Recruitment of FAM35A/derivatives GFP-fusions to a chromosomal Lac-operator array via mCherry-LacR-C20orf196. Data shown represent 3 experiments with quantifications shown in . Scale bar 10μm. c, (left and middle panel) Purified recombinant GST-FAM35A directly interacts with recombinant His-C20orf196. c, (right panel) Cell extracts expressing GFP-FAM35A/derivatives and HA-C20orf196 analysed by co-immunoprecipitation and immunoblotting. d, V5-FAM35A co-immunoprecipitates with GFP-MAD2L2; interaction with C20orf196 shown in . e, Quantification of inducible GFP-FAM35A (left panel) and GFP-C20orf196 (right panel) IRIF in γH2AX positive cells 5 h after IR (5Gy) treated with indicated siRNAs. N=4 independent experiments except (left panel) si53BP1 (n=3), siRIF1 and siMAD2L2 (n=2); and (right panel) siCTRL(n=5), siRIF1(n=3), siFAM35A(n=3). f, As in (e) but for inducible GFP-FAM35A N-terminus; n=4 independent experiments except siRIF1 (n=3). g, Endogenous MAD2L2 co-immunoprecipitates with GFP-FAM35A N-terminus. e-f, Bars represent mean ± SEM, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); individual data points plotted over bars. Statistical source data including the precise p values are shown in . All immunoblots are representative of two independent experiments; unprocessed scans of immunoblots are shown in .

a, Schematic of TRF2ts experimental setup. b, shRNA depletion of FAM35A (left panel) or C20orf196 (right panel) reduces un-capped telomere-mediated chromosome fusions. Bars represent means. The experiments were performed twice with ≥1300 chromosomes counted per condition, and individual data points plotted over bars; source data can be found in . c, FAM35Ako and C20orf196ko RPE1 cells labelled with BrdU (10μM) for 48 h then treated with 1μM camptothecin (CPT) for 1 h, pre-extracted, fixed and stained for BrdU under non-denaturing conditions to visualise ssDNA. Box and whisker plot with centre line at median, box limits at 25 th /75 th centiles and whiskers ±1.5xIQR; one-way Anova; n=3 independent experiments. d, IR-induced pRPA(S4/8) is enhanced in MEFs due to Fam35a or C20orf196 silencing. Bars represent means. The experiments were performed twice with individual data points plotted over bars; source data can be found in . e, RPE1-FAM35Ako or -C20orf196ko cells display hyper DNA-end resection (cells treated with 1μM camptothecin for 1h). Representative images from 3 independent experiments. Scale bar 10μm. f , RPE1-FAM35Ako or -C20orf196ko cells display BLM and CtIP dependent markers of excessive DNA-end resection. Box and whisker plot with centre line at median, box limits at 25 th /75 th centiles and whiskers ±1.5xIQR; one-way Anova; n=3 independent experiments. g, Enhanced BLM accrual in FAM35Ako and C20orf196ko compared with wild-type (WT) RPE1 cells fixed and stained 2 h after laser micro-irradiation. Representative images shown in left panel and quantification in right panel. Scale bar 10μm. Box and whisker plot with centre line at median, box limits at 25 th /75 th centiles and whiskers ±1.5xIQR; one-way Anova; n=3 independent experiments. For c, f and g, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); statistical source data including the precise p values can be found in .