Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Cells were treated with increasing concentrations of cisplatin for 72 hr and % survival was measured using the Crystal Violet assay. LXSN, E6, E7, E6E7 expressing cells showed respective IC50 of 2.5, 0.74, 1.27 and 0.73 uM; 72 hr. (B-D) Cells were transfected with siControl or siFancD2 for 48hrs and plated for western blotting and cisplatin survival assay. (B) Western blot showing FancD2 knockdown in the cells harvested at the time of reading survival assay. (C) Survival curves of LXSN, E6, E7 and E6E7 cells transfected with siRNA (siFancD2 or siControl) and treated with indicated doses of cisplatin for 48 hr. (D) Survival curve of siControl cells treated with indicated doses of cisplatin for 48 hrs.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Crystal Violet Assay, Expressing, Transfection, Western Blot, Clonogenic Cell Survival Assay, Knockdown

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Immunoblot showing FancD2/ FancI expression and monoubiquitination status in transduced HFK cells which were either untreated or treated with 3 uM cisplatin for 24 hr. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: Non-Ub) and total FancD2 (Ub + Non-Ub) levels are indicated beneath the corresponding lanes. (B) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells that were either untreated or treated with 3 uM cisplatin for 24 hr. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (C) Immunoblot of whole cell lysates showing levels of phosphorylated S556 FancI, total FancI, Ub-PCNA, non-Ub PCNA, and UHFR1. Vinculin acts as a loading control.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Western Blot, Expressing, Control

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Quantification of percentage of FancD2 foci co-localization with pH2AX with or without cisplatin treatment. * (p-value ≤ 0.05) and ** (p-value ≤ 0.01) denote a statistically significant difference from the similarly treated LXSN control cells. Error bars represent standard error of the mean. Quantification was based on data observed from ≥ 15 nuclei from three independent experiments. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Control, Transduction, Transfection, Expressing, Plasmid Preparation, Construct, Staining, Western Blot

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm 2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in , following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. FancD2 Ub:non-Ub ratio in cells following cisplatin withdrawal and UVB exposure was plotted alongside Figure 5B and 5C. ** (p< 0.01) denotes a statistically significant difference from 0 hr cisplatin withdrawal. ‘ns’ denotes non-significant differences. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in . Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 and total FancI in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Ubiquitin Proteomics, Western Blot, Control

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Outline of synchronization assay in HFK cells using double-thymidine block. Cells were synchronized by double-thymine block and released at various time points. Immunoblot for FancD2, FancI, cyclin A and vinculin of LXSN (B) and E6 cells (C). Cell-cycle phases at each time point were determined by flow cytometry of DNA content (D). Asynchronous (Asyn.) cells, which have not undergone thymidine block, were included for comparison.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Blocking Assay, Western Blot, Flow Cytometry, Comparison

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Immunoblot for p53 (upper panel) and RT-PCR analysis of 16E6 and GAPDH expression (lower panel) in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T). (B-C) Immunoblot showing FancD2 expression and monoubiquitination status in cells which were either untreated or treated with cisplatin (B) or nutlin (C) at 1.5 uM for 24 hr. (D-E) Cells were untreated or treated with cisplatin (E) or exposed to 10 mJ/cm 2 UVB (D) and processed similarly as in .

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transduction, Mutagenesis

Journal: bioRxiv

Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

doi: 10.1101/438945

Figure Lengend Snippet: ( A ) Scheme of the lesion ICL unhooking assay. ( B ) ICL unhooking assay gel quantified in . ( C ) Scheme of the key events during ICL repair in Xenopus egg extract and the products measured in lesion bypass assay (see and for an example of a gel and quantification. ( D ) Lesion bypass assay gel quantified in . ( E ) Repeat of the experiment shown in . ( E-H ) Efficiency of ChIP with HSF2BP, XPF, REV1 and FANCD2 antibodies from the experiment shown in . ( I ) Efficiency of MMC-induced (50 ng/ml overnight) FANCD2 monoubiquitination (appearance of the slower migrating L-form) in control and HSF2BP-overproducing (human or mouse, GFP-tagged or untagged) HeLa cells. Immunoblots with two different anti-FANCD2 antibodies are shown. Asterisks indicate non-specific bands.

Article Snippet: Antibodies used in this study were against RAD51 (rabbit 2307 ( )), FANCD2 (NB 100-316, Novus Biologicals), H2B (07-371, Millipore), MSH2 (Ab-2, Oncogene), HSP90 (ab13492, Abcam), BRCA2 (Ab-1, OP95, Calbiochem), FLAG (M2 antibody, Sigma), GFP (clones 7.1 and 13.1, Roche), ORC2 (551178,BD Pharmingen), PARP-1 (C2-10, Enzo), 6xHis tag (ab18184, Abcam), and XRCC3 (ab6494, Abcam).

Techniques: Western Blot

Journal: bioRxiv

Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

doi: 10.1101/438945

Figure Lengend Snippet: HSF2BP inhibits HR in the FA pathway ( A ) HSF2BP does not inhibit lesion unhooking in Xenopus egg extract. Prelabeled pICL was replicated in Xenopus egg extracts, replication products were isolated, digested by HincII, and separated on a denaturing agarose gel . The decline of the X-structures was quantified and plotted. See scheme in , main text and Methods for detailed description. ( B ) HSF2BP does not inhibit translesion synthesis in Xenopus egg extract. pICL was replicated in Xenopus egg extracts in the presence of 32P-lll-dCTP, replication products were isolated, digested by HincII, or HincII and SapI, and separated on a denaturing agarose gel . Extension products were quantified and plotted. Scheme of the assay in . ( C ) Inhibition of HR intermediate formation by HSF2BP revealed by 2D gel electrophoresis. pICL was replicated in Xenopus egg extracts in the presence of 32P- -dCTP, replication products were isolated, digested by HincII, and analyzed by 2D gel electrophoresis. ( D ) Stable RAD51 accumulation on pICL during repair is abrogated by HSF2BP wildtype, but not the R200T mutant. The pICL plasmid was pulled down by incubation with streptavidin beads coated with biotinylated LacI. Presence of bound RAD51 and FANCD2 was determined by immunoblotting. ( E ) Efficiency of RAD51 focus formation in HSF2BP-overproducing and control cells with or without 100 nM MMC treatment. After treatment cells were fixed, stained for immunofluorescence, mounted with DAPI and imaged using confocal microscopy. Images were scored manually for the number of foci. Data from three independent experiments is plotted, line represents average ± s.e.m., statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-test. ( F ) HSF2BP inhibits BRCA2 loading at the ICL in Xenopus egg extract. pICL replication samples at the indicated time points were analyzed by BRCA2 ChIP.

Article Snippet: Antibodies used in this study were against RAD51 (rabbit 2307 ( )), FANCD2 (NB 100-316, Novus Biologicals), H2B (07-371, Millipore), MSH2 (Ab-2, Oncogene), HSP90 (ab13492, Abcam), BRCA2 (Ab-1, OP95, Calbiochem), FLAG (M2 antibody, Sigma), GFP (clones 7.1 and 13.1, Roche), ORC2 (551178,BD Pharmingen), PARP-1 (C2-10, Enzo), 6xHis tag (ab18184, Abcam), and XRCC3 (ab6494, Abcam).

Techniques: Isolation, Agarose Gel Electrophoresis, Translesion Synthesis, Inhibition, Two-Dimensional Gel Electrophoresis, Electrophoresis, Mutagenesis, Plasmid Preparation, Incubation, Western Blot, Staining, Immunofluorescence, Confocal Microscopy

Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Co-introduction of anti-DNA ligase IV antibody reduces DNA end-joining frequency in normal diploid fibroblasts but does not affect FA cells. (A) End-joining frequency of cohesive-ended DNA was determined in HT1080 cells (black bars) and normal HDFs (white bars) in the presence of no antibody (N), in the presence of anti-DNA ligase IV antibody (L), in the presence of anti-Fancd2 antibody (D), and in the presence of both anti-DNA ligase IV and anti-Fancd2 antibodies (L + D). In all cases, antibody treatment significantly reduced plasmid end-joining levels compared to those observed in cells not treated with antibody, P < 0.0001, χ2-test. (B) End-joining frequency of cohesive-ended DNA (black bars) and blunt-ended DNA (white bars) was determined in patient-derived FA-C cells in the absence of antibody (−) and in the presence of anti-DNA ligase IV antibody (+).

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques: Plasmid Preparation, Derivative Assay

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blot analysis: 53BP1 (Bethyl Laboratories, A300-272A, 1:3000), LIN37 (Santa Cruz Biotechnology, sc-515686, 1:200), BLM (Bethyl Laboratories, A300-572A, 1:2000), BRCA1 for mouse (R and D Systems, gift from Dr. Andre Nussenzweig, NCI, 1:1000) , BRCA1 for human (Millipore Sigma, 07-434, 1:1000), RAD51 (Millipore Sigma, ABE257, 1:2000), BARD1 (Thermo Fisher Scientific, PA5-85707, 1:1000), CtIP (gift from Dr. Richard Baer, [Columbia University, New York], 1:1000), MRE11 (Novus Biologicals, NB100-142, 1:2000), RIF1 (Abcam, ab13422, 1:500), SHLD1/C20orf196 (Thermo Fisher Scientific, PA5-559280, 1:200), GAPDH (Sigma, G8795, 1:10,000), KAP1 (Genetex, GTX102226, 1:2000), FANCD2 (R and D Systems, MAB93691, 1:1000), BRCA2 for human (Proteintech, 19791-1-AP, 1:500), Rb1 (Thermo Fisher Scientific, LF-MA0173, 1:1000), Phospho-Rb (Ser780) (Cell Signaling Technology, 8180T, 1:1000), Phospho-Rb (Ser807/811) (Cell Signaling Technology, 8516T, 1:1000), PCNA (Bethyl Laboratories, A300-276A, 1:3000), CDK4: (Novus Biologicals, NBP1-31308, 1:1000), CDK4 (phosphor Thr 172) (GeneTex, GTX00778, 1:1000), and RPA (Cell Signaling Technology, 2208S, 1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy