ultra pure rnase free water  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Dimerization confers increased stability to nucleases in 5′ halves from glycine and glutamic acid tRNAs

doi: 10.1093/nar/gky495

Figure Lengend Snippet: 5′ Halves from tRNA Gly and tRNA Glu are highly stable in the non-EV fraction from MCF-7 cell conditioned medium. ( A ) MCF-7 cells were grown under defined serum-free conditions, and cell conditioned medium was collected after 48 h. The medium was split in two aliquots, one of which was immediately stored at −20°C while the other was left at 37°C overnight (ON) and then frozen. After thawing, 600 μl aliquots were concentrated 10× by ultrafiltration (using membranes with a cut-off of 5 kDa). The tRNA halves were assayed by stem-loop RT-qPCR and their quantification cycle (Cq) values are shown. Significant or not-significant (NS) changes are indicated based on two-tailed Student t test. In a parallel experiment ( B ), 40 U of murine RNase inhibitor was added to the cell conditioned medium upon collection. ( C ) The cell conditioned medium, either with or without addition of RNase inhibitor, was centrifuged at 100 000 × g for 2.5 h and the supernatant was concentrated and injected in a Superdex S75 column. Absorbance at 260 nm and 280 nm are shown in red and blue, respectively. The peak termed ‘H’ was collected, RNA was extracted and the purified RNA was either used for small RNA sequencing ( D and E ) or reinjected in the same column ( F ). Sequencing reads were collapsed to unique sequences, classified based on their origins (mature miRNAs or fragments of rRNAs, tRNAs, snoRNA, snRNAm vault RNAs or YRNAs) and relative abundances (expressed in reads per million mapped reads, RPM) were plotted against sequence length (D and E).

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Two Tailed Test, Injection, Purification, RNA Sequencing Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1093/nar/gkaa674

Figure Lengend Snippet: RNase inhibition stabilizes full-length extracellular rRNAs and tRNAs. ( A ) Size exclusion chromatography (SEC) of 100 000 × g supernatants of MCF-7 cell-conditioned medium (CM) following addition of RNase A (middle) or Ribonuclease Inhibitor (RI, bottom). Red, Blue: absorbance at 260 and 280 nm, respectively. ( B ) The earlier RI was added to the CCM during sample preparation, the higher the P0/P2 (left) and P1/P2 (right) ratios. S+, S–: serum-containing and serum-free media, respectively. SUP: supernatant. ( C ) The P0 peak was purified after SEC and treated with RNase A, which partially reconstituted the P2 peak. ( D ) Pie charts showing relative representations of different RNA biotypes in RI-SEC-seq datasets. ( E ) Size distribution of reads mapping to the 5’ half of glycine tRNA (left) or to all tRNAs (right) in the P1 peak from MCF-7 cells either with (black) or without (red) addition of RI. ( F ) Relative representation of reads mapping to different tRNA isoacceptors in the P1 peak of cells treated (top) or not (bottom) with RI. ( G ) Analysis of the P1 peak either with (+) or without (−) RI treatment (left gel) or the P0 peak (right gel) in a denaturing polyacrylamide gel. ( H ) Size distribution of small RNA sequencing reads mapping to different ncRNAs (see legend) in the P0 peak of MCF-7 cells. RPM: reads per million mapped reads. ( I ) As in (H) but showing only the reads aligning to rRNAs. Numbers indicate starting position of most reads. ( J ) Amplification of different rRNA regions by random-primed RT-qPCR in fractions collected after SEC. Gly_GSP: amplification of glycine 5’ halves using a gene-specific primer during RT. Numbers after underscores represent the 5’ position of the expected amplicon.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Inhibition, Size-exclusion Chromatography, Sample Prep, Purification, RNA Sequencing Assay, Amplification, Random Primed, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1093/nar/gkaa674

Figure Lengend Snippet: Extracellular tRNAs are processed to extracellular tRNA-derived fragments. ( A ) Schematic representation of the experimental setup used in panels B–D. ( B ) Iodixanol gradient showing most exRNAs were present in the nonvesicular fractions (RNPs) in the input sample (before cell-free processing). ( C ) Analysis of exRNAs by denaturing electrophoresis before and after the cell-free (CF) maturation step. ( D ) Northern blot analysis with probes targeting the 5’ region of different tRNAs in samples obtained as explained in (A). L: 5’ tRNA halves of 33–34 nt; S: 5’ tRNA halves of 30–31 nt. ( E ) Samples were obtained after a short (30 s) wash of U2-OS cells with HBSS+ (without RI addition), and incubated cell-free at 37°C for 0, 1 or 5 h, or for 1 h after addition of S+ medium in a 1:1 ratio. Northern blot was performed with two different probes targeting both halves of tRNA Gly GCC . ( F ) Cloverleaf representation of glycine tRNA (GCC anticodon, isodecoder #2) with red arrow showing predicted cleavage at the anticodon loop (sequence CpCpA), rendering a 33 or 34 nt 5’ fragment, and black arrows showing a putative alternative cleavage site (sequence CpCpU), generating 30–31 nt 5’ fragments. ( G ) Analysis of exRNAs in U2-OS CCM (1, 5 and 24 h, S+ medium). The concentrated nonconditioned medium was run as a control. Northern blot was done with the same probes as in panel (D), plus a 7SL RNA-specific probe. ( H ) Isopycnic centrifugation of U2-OS CCM ( t = 24 h in S+ medium). Color code (dials): green (short wash with RI; low RNase activity), yellow (endogenous RNases, released during 1 h incubation in serum-free medium without RI), red (FBS-derived RNases).

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Derivative Assay, Electrophoresis, Northern Blot, Incubation, Sequencing, Centrifugation, Activity Assay

Journal: Nucleic Acids Research

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1093/nar/gkaa674

Figure Lengend Snippet: The contents of the P0 peak can trigger dendritic cell maturation in an RNA-dependent manner. ( A ) SEC separation of the P0 and P1 peaks used for dendritic cell maturation assays. Cell-conditioned PBS was concentrated and separated into two aliquots, one of which was treated with RNase A before SEC. ( B ) Concentrated and sterilized SEC fractions were incubated with nonprimed murine bone marrow-derived dendritic cells (BMDC). The TLR3 agonist Poly (I:C) was used as a positive control. ( C ) Flow cytometry analysis of BMDC at t = 24 h post exposure to the P0 and P1 peaks (or synthetic RNAs). PI: propidium iodide. FSC: forward scatter. SSC: side scatter. Numbers to the right correspond to the percentage of viable (PI negative), CD11c-positive cells expressing high levels of class II MHC and CD80. ( D ) Percentage of matured BMDC (considered as antigen-presenting cells, APC) at t = 24 h post exposure. Triangles correspond to diluted fractions (P0: 1/100; P1: 1/100; Poly [I:C]: 1/10). E) Quantitation by ELISA of IL-1β levels in the media of BMDC analyzed by flow cytometry in the previous panel.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Incubation, Derivative Assay, Positive Control, Flow Cytometry, Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1093/nar/gkaa674

Figure Lengend Snippet: Proposed model. ( A ) Cells in culture release tRNAs, ribosomal subunits or ribosomes to the extracellular space, even outside EVs. When the CCM is analyzed by SEC, these RNAs define the P0 and P1 peaks, respectively (i). However, their detection is only possible after addition of RI to the medium. Active secretion (e.g., autophagy-dependent) might contribute to nonvesicular exRNA profiles, but damaged or dead cells with compromised plasma membrane integrity may be quantitatively more important. Extracellular RNases degrade nonvesicular RNAs and generate stable fragmentation products like glycine tRNA halves (ii), which can assemble into dimers and elute in the chromatographic P1 peak even in the absence of RI. The P2 peak is probably composed of rRNA-derived fragments (rRFs) forming tightly bound dsRNAs which are not amenable to standard small RNA sequencing techniques. While full-length tRNAs and YRNAs are not detected in the non-EV fraction in the absence of RI, those which are present inside EVs are protected from degradation. Thus, EVs are probably the only source of full-length ncRNAs in RNase-rich extracellular samples. Overall, this diagram represent the remarkable differences between what is sequenced in the extracellular space in the absence of RI and what cells actually release, as revealed by RI-SEC-seq. ( B ) A diagram explaining possible biogenetic routes for extracellular, nonvesicular tRNA Gly GCC 5’ halves.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Derivative Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: The contents of the P0 peak can trigger dendritic cell maturation in an RNA-dependent manner. A) SEC separation of the P0 and P1 peaks used for dendritic cell maturation assays. MCF-7 cells were grown in serum-free MEGM for 48 hs. The first PBS wash was discarded. Cell-conditioned PBS (t = 5 min) was concentrated and separated into two aliquots, one of which was treated with RNase A. Both samples were separated by SEC to obtain the P0 and P1 peaks (or the P0 peak from the RNase A-treated sample). B) SEC peaks were filter-sterilized and 100 µL were added to 900 µL of complete medium containing 1×10 6 nonprimed murine bone marrow-derived dendritic cells (BMDC). The TLR3 agonist Poly (I:C) was used as a positive control. C) Flow cytometry analysis of BMDC at t = 24 hours post exposure to the P0 and P1 peaks (or synthetic RNAs). PI: propidium iodide. FSC: forward scatter. SSC: side scatter. Numbers to the right correspond to the percentage of viable (PI negative), CD11c-positive cells expressing high levels of class II MHC and CD80. D) Percentage of matured BMDC (considered as antigen-presenting cells, APC) at t = 24 hours post exposure. Triangles correspond to diluted fractions (P0: 1/100; P1: 1/100; Poly [I:C]: 1/10). E) Quantitation by ELISA of IL-1β levels in the media of BMDC analyzed by flow cytometry in the previous panel.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Derivative Assay, Positive Control, Flow Cytometry, Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: Addition of ribonuclease inhibitor to cell culture conditioned medium (CCM) stabilizes extravesicular ribosomal and transfer RNAs. A) Size exclusion chromatography (SEC) of 100,000 x g supernatants of MCF-7 CCM following addition of RNase A (middle) or Ribonuclease Inhibitor (RI, bottom). Red, Blue: absorbance at 260 nm and 280 nm, respectively. B) The earlier RI was added to the CCM during sample preparation, the higher the P0 peak and the lower the P2 peak (left) and therefore the higher the P1 / P2 ratio (right). C) The P0 peak was purified after SEC and treated with RNase A, which partially reconstituted the P2 peak. D) Comparison of extracellular stabilities of the P0, P1and P2 peaks. E) Size distribution of reads mapping to the 5’ half of glycine tRNA (left) or to all tRNAs (right) in the P1 peak from MCF-7 cells either with (black) or without (red) addition of RI. F) Relative representation of reads mapping to different tRNA isoacceptors in the P1 peak of MCF-7 cells obtained after treatment (top) or without treatment (bottom) of RI. G) Analysis of the P1 peak either with (+) or without (-) RI treatment (left gel) or the P0 peak (right gel) in a denaturing (7M urea) 8% polyacrylamide gel. Sizes were estimated based on a MCF-7 RNA lysate (“cells”) and a RiboRuler Low Range small RNA ladder (left marks; the 33 nt mark was calculated based on Rf). H) Size distribution of small RNA sequencing reads mapping to rRNAs (red), tRNAs (violet) or other ncRNAs (see legend) in the P0 peak of MCF-7 cells. RPM: reads per million mapped reads. I) As in (H), but showing only the reads aligning to rRNAs. The number above each peak denotes the starting position of most reads defining that peak in the corresponding rRNA. J) Amplification by random-primed RT-qPCR of different regions of 28S, 18S and 5.8S rRNAs in different fractions collected after SEC separation of MCF-7 CCM. Gly_GSP: amplification of glycine 5’ halves by using a gene-specific primer during RT. Numbers following rRNA primers (e.g., 28S_310) represent the position of the 5’ end of the expected amplicon.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Cell Culture, Size-exclusion Chromatography, Sample Prep, Purification, RNA Sequencing Assay, Amplification, Random Primed, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: Proposed model. A) Cells in culture release tRNAs, ribosomal subunits or ribosomes to the extracellular nonvesicular space. When the CCM is analyzed by SEC, these RNAs define the P0 and P1 peaks, respectively. However, their detection is only possible after addition of RI to the medium. Regarding the mechanism responsible for the release of these RNAs, active secretion (e.g., autophagy-dependent) might contribute, but damaged or dead cells with compromised plasma membrane integrity are probably a main source of extravesicular exRNAs. Other forms of cell death can also release nucleosomes and fragmented DNA (right), although this can also occur actively by autophagy-dependent secretion. In contrast, live cells release EVs in a relatively continue fashion (center). These EVs contain ncRNAs such as tRNAs. Extracellular RNases degrade extravesicular RNAs and generate some stable fragmentation products. These products include tRNA halves, which can assemble into dimers and elute in the chromatographic P1 peak when RI is not added to the medium. We speculate that the P2 peak is composed of rRNA-derived fragments forming tightly bound dsRNAs which are not amenable to standard small RNA sequencing techniques. While full-length tRNAs and YRNAs are not detected in the non-EV fraction in the absence of RI, those which are present inside EVs are protected from degradation. Thus, EVs are probably the only source of full-length ncRNAs in RNase-rich extracellular samples. B) A diagram explaining possible biogenetic routes for extracellular, nonvesicular tRNA Gly GCC 5’ halves.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Derivative Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: Intracellular nucleic acids are released to the extracellular space after short washes in serum-free media or isotonic buffers. A) Schematic representation of the experimental protocol used in panels B-E. S(+): DMEM + 10% FBS. S(-): MEGM media, serum-free. B) RNA analysis by SEC in PBS washes #1 to #4 of MCF-7 cells. Conditions identical to those used in Figure 1 . C) An example of deconvolution analysis (Abs 260/280 ratio-to-RNA-concentration conversion) applied to a representative chromatogram of MCF-7 CCM (ultracentrifugation supernatant). D) Variation of RNA concentration corresponding to the P0 (light red) and P1 (dark red) peaks in PBS washes #1 to #4. The variation in the Abs 280 nm at the BSA peak is plotted in the right Y axis. Initial values correspond to those present in the CCM. E) Denaturing electrophoresis (7M Urea, 6% PAGE) of the concentrated P0 peak from the 4 th PBS wash of HepG2, BJ and MCF-7 cell lines. No RNA purification was performed. “Cells”: MCF-7 RNA lysate. F) U2-OS cells after incubation in serum-free DMEM plus ITS supplement for one hour. Top: monolayer. Bottom: tracking of floating nuclei (yellow line) in three consecutive shots taken one second apart from each other. G) Cluster of floating nuclei. Same conditions as in (F). H) Analysis of eIF2 alpha phosphorylation (Wblot) in nontreated (NT) MCF-7 cells, in cells exposed to four consecutive PBS washes (4 x PBS), in cells cultured after confluency (100%) or exposed to 500 µM sodium arsenite for one hour (ARS 500). Bottom: densitometry analysis in two independent biological replicates of the experiment. I) Denaturing electrophoresis (10% PAGE) of TRIzol-purified total extracellular RNA from U2-OS cells washed for 30 seconds with HBSS, ran alongside 1 µg of purified intracellular RNA from the same cell line. J) Denaturing 10% PAGE exRNA analysis in U2-OS (left) or DU 145 (right) cell-conditioned media (ITS, one hour) obtained in the presence (+) or absence (-) of 200 µM sodium arsenite. K) Denaturing electrophoresis of the purified P0 peak from E.G7-OVA cells, either treated (+) or not treated (-) with recombinant RNase-free DNase I. Sample preparation after SEC was the same as in panel (E). L) Chromatograms of cell-conditioned PBS form E.G7-OVA cells. The sample was separated into two aliquots, one of which was treated with RNase A before SEC (right). M) Proteomic analysis of the RNase-treated or not-treated (NT) P0 peak from E.G7-OVA cells. Blue: histones. Red: ribosomal proteins. NSAF: normalized spectral abundance factor. N) List of the top ten proteins from the large (left) and small (right) ribosomal subunits producing the higher number of spectra in the proteomic analysis of the P0 peak from E.G7-OVA cells.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Concentration Assay, Electrophoresis, Polyacrylamide Gel Electrophoresis, Purification, Incubation, Cell Culture, Recombinant, Sample Prep

Journal: Cell

Article Title: Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase

doi: 10.1016/j.cell.2019.04.013

Figure Lengend Snippet: 90A RNP Recognition by the Pan2 RNase Domain and the Pan3straight Pseudokinase Domain (A) Views of the interaction between UCH-like-RNase modules of Pan2 (violet and pink, respectively) and the RRM1-RRM2 module of the first Pab1 (dark green). The pseudo-atomic model (right panel) is superposed on the cryo-EM density (central panel). The left panel identifies the overall position of the interface in the context of the reconstruction (shown as segmented density, as in Figure 3 B). Difference density for the RNA is shown in mesh representation, in black. The directionality of RNase-Pab1 recognition is fixed by the defined polarity of the poly(A) recognition by RRM1-RRM2 (3′ end at the N terminus, 5′ end at the C terminus) as well as the 3′-to-5′ exonuclease activity of Pan2 (additional details in Figure S5 A). (B) Corresponding views of the interaction between the Pan3s pseudokinase domain and the first RNP oligomerization interface (i.e., RRM4-linker helix of the first Pab1 protomer and the RRM1-RRM2 module of the second protomer). Pan3s is shown in a surface representation colored according to evolutionary conservation (dark orange for conserved residues). The RNP contacts the connecting segment (CS) of Pan2, also shown in a surface representation and colored according to conservation (conserved residues in dark blue; see also Figure S5 B).

Article Snippet: Pellets were resuspended in 40 μl RNase free water and 800 μg total RNA from each strain were 3′-radioactive labeling by 5′-32P pCp using T4 RNA ligase 1 (NEB) for 1 h at 37°C.

Techniques: Cryo-EM Sample Prep, Activity Assay

Journal: Cell

Article Title: Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase

doi: 10.1016/j.cell.2019.04.013

Figure Lengend Snippet: Determinants of Pan2-Pan3 Recruitment to the Poly(A)/RNP, Related to Figure 5 (A) Details of the interactions between the second Pab1-Pab1 oligomerization interface of the 90A RNP and the Pan2 WD40 domain. The panel on the left highlights the overall position of the interface in the context of the reconstruction (shown as segmented density, as in Figure 3 B). Conserved surface residues of the WD40 domain in proximity to the Pab1-Pab1 interface are accentuated in a dark blue shade in the surface representation (panel in the middle) and as spheres in the cartoon model on the right. (B) Pan2ΔWD40-Pan3 deadenylation activity is not stimulated by Pab1. 5′ radioactively labeled model-90A RNA was mixed with Pab1 (in a 1:3 RNA:protein ratio,”+Pab1”) and incubated with either wild-type Pan2-Pan3 (left hand side of the gel) or Pan2ΔWD40-Pan3 (right hand side of the gel, equimolar and 10x the amount of wild-type Pan2-Pan3) over a 2 h period. The model-90A RNA in the absence of Pab1 (“no Pab1”) was in parallel also used as substrate in similar deadenylation reactions. At indicated time points samples were taken and analyzed on a 6% Urea-PAGE followed by phosphorimaging. (C) The Pan2 WD40 domain influences poly(A) tail length in vivo . The upper panel shows phosphorimages of 8% UREA-PAGE of the pCp labeled, RNase A treated poly(A) isolations from mutant Pan2 yeast strains (Pan2-EGFP tagged strains on the left, S. cerevisiae strains carrying untagged Pan2 variants on the right). In the bottom panel is the respective anti-EGFP western blot. (D) Removal of the Pan3 N terminus has a limited effect on deadenylation activity of a yeast 90A RNP. 5′ radioactively labeled model-90A RNA was mixed with Pab1 (in a 1:3 RNA:protein ratio) and incubated with either wild-type Pan2-Pan3 (left hand side of the gel) or Pan2-Pan3Δ (1–138) (right hand side of the gel) over a 2 h period. At indicated time points samples were taken and analyzed on a 6% Urea-PAGE followed by phosphorimaging. (E) The Pan3 N-terminal Zn-fingers do not contribute to the poly(A) length dependency of the Pan2-Pan3–poly(A)/Pab1 interaction but increase affinity for the poly(A)/Pab1 RNP in co-precipitation experiments. Pan2cat-Pan3, Pan2cat-Pan3Zn-mut (in which the three Cys and one His coordinating the Zn ion have been mutated to Ser or Ala respectively) as well as Pan2cat-Pan3ΔZn (which is Pan2cat-Pan3Δ[1-74]) were preys in a Strep-Tactin pull-down with 30A/Pab1-Strep and 60A/Pab1-Strep as bait. The eluate off the Strep-Tactin resin was analysed on a 4%–12% SDS-PAGE followed by Coomassie staining.

Article Snippet: Pellets were resuspended in 40 μl RNase free water and 800 μg total RNA from each strain were 3′-radioactive labeling by 5′-32P pCp using T4 RNA ligase 1 (NEB) for 1 h at 37°C.

Techniques: Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, In Vivo, Mutagenesis, Western Blot, SDS Page, Staining