Journal: Molecular cell

Article Title: Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF

doi: 10.1016/j.molcel.2019.07.039

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA Polymerase I, Large (Klenow) Fragment , New England Biolabs , Cat. # M0210.

Techniques: In Vitro, RNA Binding Assay, Recombinant, Staining, Flow Cytometry, Sample Prep, Expressing, Imaging, Knock-In, Plasmid Preparation, Software

Journal: Journal of Clinical Medicine

Article Title: Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies

doi: 10.3390/jcm10102128

Figure Lengend Snippet: SARS-CoV-2 (RBD) IgG and PRNT antibody titers in follow-up serum samples of six individuals vaccinated with BNT162b2. neg. = negative; - not tested.

Article Snippet: In a large study conducted in Israel, the recently available BNT162b2 mRNA Covid-19 Vaccine (BioNTech/Pfizer) reduced the risk of symptomatic COVID-19 disease by 94% and the risk of asymptomatic SARS-CoV-2 infection by 90% [ ].

Techniques:

Journal: Nucleic Acids Research

Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

doi: 10.1093/nar/gkaa516

Figure Lengend Snippet: Assessment of interference by rNTPs. ( A–D ) The assay reactions (197-nt templates) were spiked with the indicated amounts of rNTPs and the change in the dNTP signal was measured. The concentrations shown are final reaction concentrations. The estimated upper ranges for rNTP/dNTP ratios are shown for cultured cell and post-mitotic tissues. (E, F) The effect of reaction time on the apparent interfering GTP ( E ) and ATP ( F ) signal. The purple dashed line represents the expected increase in the interfering signal assuming true ATP incorporation, essentially steady-state supply of ATP, and a time-dependent exhaustion of dATP during the reaction. Reaction time-independent increase in signal suggests trace amounts (∼0.015%) of dATP in the ATP preparation. (G, H) Melt curve analysis of RNAse HII-nicked 50-nt products of rGTP/dGTP ( G ) and rATP/dATP assay reactions ( H ). For reactions containing 2 mM ATP, 1.6 mM MgCl 2 was added to compensate the chelation of Mg 2+ by ATP. (I, J) Denaturing polyacrylamide (13%) gel electrophoresis of the reaction products with and without thermostable RNAse HII. The rNTP-incorporating Thermococcus 9°N-7 mutant DNA polymerase (Therminator) was used as positive control for the ATP incorporation. shows similar assessment of ATP incorporation using alkaline cleavage of ribonucleotide bonds. The error bars in the figures represent standard deviation and mean from three technical replicates.

Article Snippet: As a positive control for rNTP incorporation, a Thermococcus 9°N-7 mutant (D141A/E143A/A485L) DNA-RNA polymerase (available commercially as Therminator polymerase from New England Biolabs) readily incorporated ATP and gave a prominent cleavage product, which confirmed that the RNAse HII did cut this particular ribonucleotide bond.

Techniques: Cell Culture, Nucleic Acid Electrophoresis, Mutagenesis, Positive Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

doi: 10.1093/nar/gkaa516

Figure Lengend Snippet: Utilization of thermostable RNAse HII and differential DNA melting temperature to eliminate interfering rNTP signal. ( A ) Schematic presentation of an assay modification to remove interfering rNTP signal. ( B ) Temperature-dependent decrease in interfering GTP signal in reactions containing 0.2 U/ml thermostable RNAse HII. Shaded grey area illustrates the optimal temperature range for the recoding of end-point fluorescence. ( C ) Apparent increase in dNTP signal in the presence of excess rNTP when the rNTP elimination modification was used. The bar graphs represent mean and standard deviation from three replicates.

Article Snippet: As a positive control for rNTP incorporation, a Thermococcus 9°N-7 mutant (D141A/E143A/A485L) DNA-RNA polymerase (available commercially as Therminator polymerase from New England Biolabs) readily incorporated ATP and gave a prominent cleavage product, which confirmed that the RNAse HII did cut this particular ribonucleotide bond.

Techniques: Modification, Fluorescence, Standard Deviation

Journal: Aging Cell

Article Title: The variant senescence‐associated secretory phenotype induced by centrosome amplification constitutes a pathway that activates hypoxia‐inducible factor ‐1α

doi: 10.1111/acel.13766

Figure Lengend Snippet: Centrosome amplification triggers a variant senescence‐associated secretory phenotype (SASP). (a) Experimental scheme for the matrigel‐coated transwell invasion experiment. Transwell inserts physically separate mCherry MDA‐MB468 cells (top chamber) from the indicated MCF10A cells (bottom chamber). Paracrine invasion is scored from the number of mCherry MDA‐MB468 cells that migrate to the bottom chamber. (b, c) MCF10A cells with centrosome amplification promote the invasion of MDA‐MB468 mCherry cells. Representative images (b) and fold increase (c) in mCherry MDA‐MB468 cells that crossed the matrigel‐coated transwell upon co‐culturing with the indicated MCF10A cells. (d) Centrosome amplification alters expression of genes related to cell motility and senescence. Ingenuity Pathway Analysis (IPA) of gene expression changes in MCF10A cells with centrosome amplification relative to controls revealing the top pathways altered by centrosome amplification. *Hepatic Fibrosis/ Hepatic Stellate Cell Activation is a senescence‐regulated process (Krizhanovsky et al., ). (e–h) Induction of senescence‐related gene expression in cells with centrosome amplification. Gene set enrichment analysis (GSEA) revealed strong enrichment of genes upregulated (e, g) or downregulated (f, h) with senescence in MCF10A cells with centrosome amplification (e, f) and RPE‐1 cells with centrosome amplification (g, h) relative to controls. NES: normalised enrichment score; FDR: false discovery rate. (i, j) Induction of secreted protein expression in cells with centrosome amplification. Gene set enrichment analysis (GSEA) revealed enrichment of genes annotated to the extracellular region in MCF10A cells with centrosome amplification (i) or RPE‐1 cells with centrosome amplification (j). NES: normalised enrichment score; FDR: false discovery rate. (k, l) Induction of senescence‐related gene expression in cells with centrosome amplification. Gene set enrichment analysis (GSEA) revealed strong enrichment of genes upregulated (k) or downregulated (l) with senescence in RPE‐1 tetraploids relative to “evolved tetraploids.” NES: normalised enrichment score; FDR: false discovery rate. (m, n) Proliferation arrest after centrosome amplification. Representative images (m) and quantification (n) of cells that cycled through S‐phase (24 hr. EdU‐label, red) and DAPI (blue) in MCF10A cells with (PLK4) and without (608) centrosome amplification. (o) Centrosome amplification induces retinoblastoma protein phosphorylation. Rb, Phospho Rb S780 and GAPDH (loading control) immunoblots of lysates from MCF10A cells with and without centrosome amplification. (p, q) Centrosome amplification increases cell size. Suspended (trypsinised) (p) and adherent (q) cell size measured for MCF10A cells with or without centrosome amplification. (r, s) Increased SA‐β‐Gal staining in cells with centrosome amplification. Representative images (r) and quantification (s) of SA‐β‐Gal (blue) staining of MCF10A cells with and without centrosome amplification or positive control with doxorubicin treatment. (t, u) Centrosome amplification does not induce DNA damage. Representative images (t) and quantification (u) of γ H2AX foci (green) in MCF10A cells with (PLK4) and without (608) centrosome amplification as compared to DNA damage from doxorubicin treatment. Cells in S‐phase are labelled with Edu (red) pulse and excluded from the quantification. (v, w, x) Centrosome amplification does not induce paracrine invasion. (v) Experimental scheme for the paracrine senescence assay. Transwell inserts ensure physical separation between MCF10A PLK4(top), 608(top) or wild‐type cells (bottom). Representative images (w) and quantification (x) of SA‐β‐Gal (blue) staining of MCF10A wild‐type cells that were co‐cultured with PLK4 or 608 or doxorubicin treated cells. (y, z) RNAi‐mediated knockdown of p53 or LATS2 releases MCF10A cells with centrosome amplification from proliferation arrest. (y) Quantification of control, p53 knockdown and LATS2 knockdown MCF10A cells with (PLK4) and without (608) centrosome amplification, which cycled through S‐phase (24 hr EdU‐label, red). (z) p53, LATS2 and GAPDH (loading control) immunoblots of lysates from control 608, p53 KD 608, LATS2 KD 608, control PLK4, p53 KD PLK4 and LATS2 KD PLK4 cells. Scale bar, 50 μm. All data are means ± SEM from n = 3 independent experiments, ** p < 0.01, **** p < 0.0001; analysed with Student's t test except m, which was with one‐way ANOVA, Tukey's multiple comparison test. Scale bars, 50 μm.

Article Snippet: The ANGPTL4 mouse antibody mAb11F6C4, used at 40 μg/ml for blocking experiments, was a gift from Tan Nguan Soon, Andrew (Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore). siRNA against CYBA p22phox (Santa Cruz), smartpool siRNA against LATS2 and p53 (Dharmacon).

Techniques: Amplification, Variant Assay, Expressing, Gene Expression, Activation Assay, Phospho-proteomics, Control, Western Blot, Staining, Positive Control, Cell Culture, Knockdown, Comparison

Journal: Aging Cell

Article Title: The variant senescence‐associated secretory phenotype induced by centrosome amplification constitutes a pathway that activates hypoxia‐inducible factor ‐1α

doi: 10.1111/acel.13766

Figure Lengend Snippet: Centrosome amplification‐induced ROS. (a, b) RNAi‐mediated knockdown of p53 or LATS2 does not affect nuclear HIF‐1α accumulation after centrosome amplification. Shown are representative images (a) and quantification (b) of nuclear HIF‐1α levels in the indicated cells. (c) Small molecule Rac‐1 inhibition prevents the accumulation of nuclear HIF‐1α after centrosome amplification. The indicated MCF10A cells were treated with 50 μm NSC23766 or vehicle and HIF‐1α nuclear accumulation was measured. (d, e) CRISPR‐mediated gene disruption of TRIO blocks nuclear HIF‐1α accumulation after centrosome amplification. Gene targeting of a pool of cells was performed in the indicated MCF10A cells prior to the initiation of centrosome amplification. Shown are representative image (d) and quantification (e) of nuclear HIF‐1α levels in the indicated cells. (f) siRNA knockdown of p22 phox inhibits nuclear HIF‐1α accumulation after centrosome amplification. (g) Trio is required for the upregulation of HIF‐1α‐induced genes in MCF10A cells with centrosome amplification. Shown is a GSEA plot comparing cells with centrosome amplification with or without TRIO gene disruption. (h) The induction of ANGPTL4 by centrosome amplification requires Trio and ROS. Shown is the fold induction of ANGPTL4 from RNA‐Seq after centrosome amplification in MCF10A cells after the indicated treatments. (i) Accumulation of superoxide after centrosome amplification. Superoxide levels were measured by dihydroethidium labelling in MCF10A cells with and without centrosome amplification. Pyocyanin treatment is the positive control. (j) Conversion of superoxide into hydrogen peroxide further induces nuclear HIF‐1α accumulation in cells with centrosome amplification. Shown are the fold changes in nuclear HIF‐1α in the indicated MCF10A cells after treatment with TEMPOL. (k, l) Catalase blocks the nuclear accumulation of HIF‐1α in cells with centrosome amplification. Representative images (k) and quantification (l) of HIF‐1α in the indicated MCF10A cells with and without catalase medium addition. (m) GSEA showing that catalase treatment prevents the upregulation of the custom HIF‐1α signature up gene set in MCF10A cells. All data are means ± SEM from n = 3 independent experiments, * p < 0.05, ** p < 0:01, *** p < 0:001, **** p < 0:0001; analysed with one‐way ANOVA, Tukey's multiple comparison test. Scale bars, 50 μm. (n) Model for centrosome amplification‐induced SASP.

Article Snippet: The ANGPTL4 mouse antibody mAb11F6C4, used at 40 μg/ml for blocking experiments, was a gift from Tan Nguan Soon, Andrew (Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore). siRNA against CYBA p22phox (Santa Cruz), smartpool siRNA against LATS2 and p53 (Dharmacon).

Techniques: Amplification, Knockdown, Inhibition, CRISPR, Disruption, RNA Sequencing, Positive Control, Comparison

Journal: Molecular cell

Article Title: HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

doi: 10.1016/j.molcel.2012.09.017

Figure Lengend Snippet: Figure 2. Contribution of SLC7A5 Expression to HIF2a-Dependent mTORC1 Activation (A) Left panel, relative Slc7a5 gene expression in HIF2a (P-A)2, HIF2a (P-A)2bHLH*, HIF1a (P-A)2, and WT8 cells. The mean is shown, and error bars represent SEM (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001). Right panel, relative bNIP3 gene expression in HIF2a (P-A)2, HIF1a (P-A)2, and WT8 control cells (n = 4). The mean is shown; error bars represent SEM (n = 4, *p < 0.05, ***p < 0.001). (B) Western blot analyses of SLC7A5 and tubulin protein levels in HIF2a (P-A)2, HIF2a (P-A)2bHLH*, and WT8 control cells. (C) WT8 control cells were transfected with scrambled control small interfering RNA (siSCR), and HIF2a (P-A)2 WT8 cells were transfected with either siSCR or siRNA against Slc7a5 (siSLC7A5). The cells were subsequently cultured for 72 hr in media with 50% of the normal amino acid content. Whole-cell extracts were analyzed by western blot with antibodies indicated. See also Figure S2.

Article Snippet: SLC7A5 (sc-62555-V) and control (sc-108080) small hairpin RNA (shRNA) lentiviral particles (from Santa Cruz Biotechnology) were used to generate stable transfectants in the 786-O cells, 786-O-shSLC7A5 cells, and their corresponding control 786-O-shSCR cells.

Techniques: Expressing, Activation Assay, Gene Expression, Control, Western Blot, Transfection, Small Interfering RNA, Cell Culture

Journal: Molecular cell

Article Title: HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

doi: 10.1016/j.molcel.2012.09.017

Figure Lengend Snippet: Figure 3. Effect of Endogenous HIF2a on mTORC1 Activity and SLC7A5 Expression in 786-O VHL-Deficient Cells Western blot analysis with antibodies as shown in (A) HIF2a-silenced 786-O cells and their corresponding control siSCR-transfected 786-O cells 72 hr after transfection, (B) 786-O and their counterparts in which VHL expression was restored (786-O-VHL), and (C) SLC7A5-silenced 786-O cells and their corre- sponding control 786-O-shSCR cells. All these experiments were performed by culturing 786-O cells for 48 hr in media with 5% of the normal content of essential amino acids and glutamine.

Article Snippet: SLC7A5 (sc-62555-V) and control (sc-108080) small hairpin RNA (shRNA) lentiviral particles (from Santa Cruz Biotechnology) were used to generate stable transfectants in the 786-O cells, 786-O-shSLC7A5 cells, and their corresponding control 786-O-shSCR cells.

Techniques: Activity Assay, Expressing, Western Blot, Control, Transfection

Journal: Molecular cell

Article Title: HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

doi: 10.1016/j.molcel.2012.09.017

Figure Lengend Snippet: Figure 4. HIF2a Binds to the Slc7a5 Prox- imal Promoter (A) Schematic representation of the human Slc7a5 gene indicating the positions of the proximal promoter and intron 1 and the nucleotide se- quences corresponding to the putative hypoxia- response elements highlighted in bold. (B and C) ChIP assay to assess the relative HIF2a binding activity to the human Slc7a5 proximal promoter or intron 1 in (B) HIF2a (P-A)2, HIF2a (P-A)2bHLH*, and WT8 control cells or (C) 786-O and their counterparts in which VHL expression was restored (786-O-VHL). Representative gel showing DNA amplified in the ChIP assays is shown. The mean is shown; error bars represent SEM (n = 5, *p < 0.05). IgG, immunoglobulin G. See also Figure S3.

Article Snippet: SLC7A5 (sc-62555-V) and control (sc-108080) small hairpin RNA (shRNA) lentiviral particles (from Santa Cruz Biotechnology) were used to generate stable transfectants in the 786-O cells, 786-O-shSLC7A5 cells, and their corresponding control 786-O-shSCR cells.

Techniques: Binding Assay, Activity Assay, Control, Expressing

Journal: Molecular cell

Article Title: HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

doi: 10.1016/j.molcel.2012.09.017

Figure Lengend Snippet: Figure 5. Role of the HIF2a-Dependent SLC7A5 Pathway in Cell Proliferation and Xenograft Growth (A) Cell number fold induction in control, HIF2a (P-A)2, and HIF2a (P-A)2bHLH* WT8 cells cultured for 72 hr in normal media (left panel) or in media containing 5% of the normal amino acid concen- tration (right panel). Statistical significance was indicated only when HIF2a (P-A)2 proliferation differed significantly from that of both HIF2a (P-A)2bHLH* WT8 and control WT8 cells. The mean is shown; error bars represent SEM (n = 4, *p < 0.05, **p < 0.01. (B) WT8 control cells were transfected with scrambled control siRNA (siSCR), and HIF2a (P-A)2 WT8 cells were transfected with either siSCR or siRNA for Slc7a5 (siSLC7A5). Twenty- four hours after transfection, cells were seeded at the same cell density in media containing 5% of the regular amino acid concentration, and the increase in cell number was analyzed after 72 hr. The mean is shown; error bars represent SEM (n = 4, *p < 0.05, ***p < 0.001). (C) WT8 control cells and HIF2a (P-A)2 WT8 cells were treated with rapamycin (20 nM) and cultured in media containing 5% of the regular amino acid concentration. The increase in cell number was analyzed after 72 hr. The mean is shown; error bars represent SEM (n = 8, **p < 0.01). (D) Tumor-volume evolution (Vol) over a 45 day period of SLC7A5-silenced 786-O cells (white squares) and their corresponding control 786-O- shSCR cells (black squares) injected subcutane- ously in the dorsal flanks of immunosuppressed severe combined immunodeficiency mice, as detailed in the Experimental Procedures section. The mean is shown; error bars represent SEM (n = 18, *p < 0.05, **p < 0.01). See also Figure S4.

Article Snippet: SLC7A5 (sc-62555-V) and control (sc-108080) small hairpin RNA (shRNA) lentiviral particles (from Santa Cruz Biotechnology) were used to generate stable transfectants in the 786-O cells, 786-O-shSLC7A5 cells, and their corresponding control 786-O-shSCR cells.

Techniques: Control, Cell Culture, Transfection, Concentration Assay, Injection

Journal: Molecular cell

Article Title: HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

doi: 10.1016/j.molcel.2012.09.017

Figure Lengend Snippet: Figure 6. Hypoxia and HIF2a-Dependent mTORC1 Activity and Slc7a5 Expression in Lung Tissue (A) Wild-type mice were exposed to hypoxia (10% O2) (Hx10%) or normoxia (N) for 4 days. Protein extracts from the lungs were analyzed by western blots with antibodies as shown. (B) Lung phospho-rpS6Ser235/6 immunostaining of mice exposed to normoxia or hypoxia (10% O2) for 4 days and in Vhlfl-UBC-Cre-ERT2, VhlflHIF2afl- UBC-Cre-ERT2, and the corresponding control mice. Quantification of the number of bronchial epithelial cells positive for phospho-rpS6Ser235/6

Article Snippet: SLC7A5 (sc-62555-V) and control (sc-108080) small hairpin RNA (shRNA) lentiviral particles (from Santa Cruz Biotechnology) were used to generate stable transfectants in the 786-O cells, 786-O-shSLC7A5 cells, and their corresponding control 786-O-shSCR cells.

Techniques: Activity Assay, Expressing, Western Blot, Immunostaining, Control

Journal: Molecular cell

Article Title: HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

doi: 10.1016/j.molcel.2012.09.017

Figure Lengend Snippet: Figure 7. HIF2a-Dependent mTORC1 Activ- ity and SLC7A5 Expression in VHL-Deficient Liver Tissue (A) Protein extracts from the livers of Vhlfl-UBC- Cre-ERT2, VhlflHIF2afl-UBC-Cre-ERT2, and the corresponding control mice were analyzed by western blots with the indicated antibodies. (B) SLC7A5 immunostaining of liver tissue (hepa- tocytes) from Vhlfl-UBC-Cre-ERT2, VhlflHIF2afl- UBC-Cre-ERT2, and corresponding control mice. Relative expression of Slc7a5 and Pgk-1 genes in the livers of Vhlfl-UBC-Cre-ERT2 (n = 7), VhlflHIF2afl-UBC-Cre-ERT2 (n = 6), and corre- sponding control mice (n = 7). The mean is shown; error bars represent SEM (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S6.

Article Snippet: SLC7A5 (sc-62555-V) and control (sc-108080) small hairpin RNA (shRNA) lentiviral particles (from Santa Cruz Biotechnology) were used to generate stable transfectants in the 786-O cells, 786-O-shSLC7A5 cells, and their corresponding control 786-O-shSCR cells.

Techniques: Expressing, Control, Western Blot, Immunostaining

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 3. Microscopic image and growth curve of HEK293 WT and RH2A-KO cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 5. Expression of RH2A.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Expressing

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 6. Expression of RH2A variants with AGS- causing mutation in RH2A-KO cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Expressing, Mutagenesis