Journal: Cell reports

Article Title: The polySUMOylation axis promotes nucleolar release of Tof2 for mitotic exit

doi: 10.1016/j.celrep.2024.114492

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-Smt3 , Santa Cruz , Cat# SC-137158; RRID:AB_201891.

Techniques: Recombinant, Protease Inhibitor, Gel Extraction, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

doi: 10.1371/journal.pone.0144508

Figure Lengend Snippet: (A) The diagram shows that compared to NPCs in the nuclear envelope, ALPCs are embedded in the membrane cisternae of annulate lamellae that are often connected to the membrane network of ER. (B) Human cervical cancer cells (HeLa) were double-labeled with anti-RanGAP1 antibody and anti-SUMO1 mAb (21C7) or mAb414 for staining NPCs and ALPCs and then analyzed by immunofluorescence microscopy. (C) Mouse embryonic fibroblasts (NIH3T3) were double-stained with anti-RanGAP1 antibody and mAb414 or anti-RanBP2 mAb. (D) Rat primary cortical/hippocampal neurons (PN) were double-labeled with anti-RanGAP1 antibody and mAb414. (E) Human bronchial/tracheal smooth muscle cells (SMC) cells were double-stained with anti-RanGAP1 antibody and mAb414 or anti-RanBP2 mAb. Bar, 10 μm. The boxes at the top corner of each image show an enlarged version of inlets. (F) Annulate lamellae are highly abundant in SMC cells. 60 SMC cells were double-stained with anti-RanGAP1 antibody and mAb414. All the ALPC foci in each cell were counted under Olympus inverted IX81 fluorescence microscope using Z-stacks. The number of ALPC foci per cell was classified into three categories (10–50, 50–100 and ≥100), and the percentage of cells in each category was indicated. Each column represents the mean value ± SEM ( N = 60) (ALPC foci/cell: 10–182; Average = 63).

Article Snippet: Antibodies used in this study were obtained from the following sources: anti-RanGAP1 (19C7) and anti-SUMO1 (21C7) mouse monoclonal antibodies (mAbs) [ ], Dr. Michael Matunis (Johns Hopkins, Baltimore, MD); anti-RanGAP1 rabbit polyclonal antibody (pAb) [ ], Dr. Mary Dasso (NIH, Bethesda, MD); anti-POM121 rabbit pAb (EMD Millipore); anti-tubulin mouse mAb (Sigma); anti-Myc (9E10) mAb (Santa Cruz); anti-lamin B goat pAb (Santa Cruz); anti-nucleophosmin rabbit pAb (Santa Cruz); anti-RanBP2 mouse mAb (Santa Cruz); anti-RanBP2 rabbit pAb (Abcam); mAb414 mouse mAb (Covance); anti-Myc rabbit pAb (Cell Signaling); anti-calreticulin rabbit pAb (Calbiochem); anti-ELYS mouse mAb (Bio Matrix Research); anti-ELYS rabbit pAb (Bethyl Laboratories); anti-importin α mouse mAb (BD Biosciences); anti-importin β mouse mAb (BD Biosciences); anti-CRM1 mouse mAbs (BD Biosciences; Santa Cruz); anti-FLAG mouse mAb (M2) (Sigma); anti-FLAG rabbit pAb (Sigma); anti-Ubc9 rabbit pAb (Abcam); anti-Ran mouse mAb (BD Biosciences).

Techniques: Membrane, Labeling, Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

doi: 10.1371/journal.pone.0144508

Figure Lengend Snippet: (A-D) HeLa cells were double stained with mAb414 and calreticulin antibody for labeling ER network (A), mAb414 and RanBP2 antibody (B), tubulin and calreticulin antibodies (C), and tubulin and RanBP2 antibodies (D) followed by immunofluorescence microscopy. The enlarged versions of inlets are shown at the bottom or top corner of each image (A-D). The arrows indicate the positions of the ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes that are most distant from the corresponding nucleus (D). The immunofluorescent images were taken using Olympus inverted IX81 widefield fluorescence microscope with U-Plan S-Apo 60×/1.35 NA oil immersion objective. Bar, 10 μm.

Article Snippet: Antibodies used in this study were obtained from the following sources: anti-RanGAP1 (19C7) and anti-SUMO1 (21C7) mouse monoclonal antibodies (mAbs) [ ], Dr. Michael Matunis (Johns Hopkins, Baltimore, MD); anti-RanGAP1 rabbit polyclonal antibody (pAb) [ ], Dr. Mary Dasso (NIH, Bethesda, MD); anti-POM121 rabbit pAb (EMD Millipore); anti-tubulin mouse mAb (Sigma); anti-Myc (9E10) mAb (Santa Cruz); anti-lamin B goat pAb (Santa Cruz); anti-nucleophosmin rabbit pAb (Santa Cruz); anti-RanBP2 mouse mAb (Santa Cruz); anti-RanBP2 rabbit pAb (Abcam); mAb414 mouse mAb (Covance); anti-Myc rabbit pAb (Cell Signaling); anti-calreticulin rabbit pAb (Calbiochem); anti-ELYS mouse mAb (Bio Matrix Research); anti-ELYS rabbit pAb (Bethyl Laboratories); anti-importin α mouse mAb (BD Biosciences); anti-importin β mouse mAb (BD Biosciences); anti-CRM1 mouse mAbs (BD Biosciences; Santa Cruz); anti-FLAG mouse mAb (M2) (Sigma); anti-FLAG rabbit pAb (Sigma); anti-Ubc9 rabbit pAb (Abcam); anti-Ran mouse mAb (BD Biosciences).

Techniques: Staining, Labeling, Immunofluorescence, Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

doi: 10.1371/journal.pone.0144508

Figure Lengend Snippet: ALPCs may serve as the docking or assembling sites for importin α/β-mediated import complexes followed by their dissociation for nuclear import. On the other hand, the ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes may function in the disassembly of CRM1-mediated export complexes by mediating RanGTP hydrolysis.

Article Snippet: Antibodies used in this study were obtained from the following sources: anti-RanGAP1 (19C7) and anti-SUMO1 (21C7) mouse monoclonal antibodies (mAbs) [ ], Dr. Michael Matunis (Johns Hopkins, Baltimore, MD); anti-RanGAP1 rabbit polyclonal antibody (pAb) [ ], Dr. Mary Dasso (NIH, Bethesda, MD); anti-POM121 rabbit pAb (EMD Millipore); anti-tubulin mouse mAb (Sigma); anti-Myc (9E10) mAb (Santa Cruz); anti-lamin B goat pAb (Santa Cruz); anti-nucleophosmin rabbit pAb (Santa Cruz); anti-RanBP2 mouse mAb (Santa Cruz); anti-RanBP2 rabbit pAb (Abcam); mAb414 mouse mAb (Covance); anti-Myc rabbit pAb (Cell Signaling); anti-calreticulin rabbit pAb (Calbiochem); anti-ELYS mouse mAb (Bio Matrix Research); anti-ELYS rabbit pAb (Bethyl Laboratories); anti-importin α mouse mAb (BD Biosciences); anti-importin β mouse mAb (BD Biosciences); anti-CRM1 mouse mAbs (BD Biosciences; Santa Cruz); anti-FLAG mouse mAb (M2) (Sigma); anti-FLAG rabbit pAb (Sigma); anti-Ubc9 rabbit pAb (Abcam); anti-Ran mouse mAb (BD Biosciences).

Techniques:

Journal:

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

doi: 10.1093/emboj/cdf510

Figure Lengend Snippet: Fig. 1. Sp3 is SUMO modified in vivo. (A) Schematic representation of HA- and FLAG-tagged wild-type Sp3 protein Sp3WT and the Sp3 mutant Sp3SD expressed in SL2 cells (Braun and Suske, 1999) and in MEFs deficient of endogenous Sp3 (H.Göllner and G.Suske, unpublished data). Grey boxes indicate the two glutamine-rich activation domains A and B, and three black stripes the zinc fingers of the DNA-binding domain (DBD) of Sp3. The ID of Sp3 is depicted by hatched stripes. Amino acids that are deleted in the Sp3SD mutant protein are shown. The single lysine within this sequence is underlined. (B) Western blot analyses of epitope-tagged Sp3WT and mutant Sp3SD protein. HA- and FLAG-tagged Sp3WT- or Sp3SD-expressing SL2 cells (lanes 2 and 3) and MEFs (lanes 4, 5 and 6) were lysed with SDS-containing buffer. Proteins were separated on 7.5% SDS– polyacrylamide gels and blotted to PVDF membranes. Membranes were subsequently incubated with HA- (αHA) or Sp3-specific (αSp3c) antibodies as indicated. Arrows point to the covalently modified wild-type Sp3WT protein. Asterisks depict C-terminally deleted degradation products of Sp3 and Sp3SD that were detectable with the αHA antiserum but not with the αSp3c antibodies that recognize an epitope at the Sp3 C-terminal end. Lane 1 contains affinity-purified epitope-tagged Sp3 protein (Sp3pur.) (Braun et al., 2001) lacking the covalent modification. (C) GFP fusion vectors (3 µg) for GFP–Sp3WT, GFP–Sp3K/R, GFP–SUMO-1 and GFP–SUMO-2 were transiently transfected in Ishikawa cells as indicated. Cells were lysed and equal amounts of total cellular proteins (20 µg per lane) were separated on a 6.0% SDS–polyacrylamide gel and blotted to PVDF membranes. Detection was by immunoblotting with αGFP antibodies. The arrows point to SUMO-modified GFP–Sp3WT. The occurrence of two GFP–SUMO-2 forms is most likely due to incomplete processing.

Article Snippet: The following antibodies and dilutions (in 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20, 1% skim milk) were used: mouse anti-GFP (Clontech) 1:2000; rabbit anti-Sp3 polyclonal IgG (Santa Cruz) 1:2000; rabbit anti-Sp3 serum ( Hagen et al ., 1994 ) 1:1000; rabbit anti-HA (Santa Cruz) 1:2000; rabbit anti-SUMO-1 (Santa Cruz) 1:1000; horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science) 1:10 000; and horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science) 1:10 000.

Techniques: Modification, In Vivo, Mutagenesis, Activation Assay, Zinc-Fingers, Binding Assay, Sequencing, Western Blot, Expressing, Incubation, Affinity Purification, Transfection

Journal:

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

doi: 10.1093/emboj/cdf510

Figure Lengend Snippet: Fig. 2. In vitro SUMOylation and deSUMOylation of Sp3 fragments. (A) Schematic drawing of the conjugation pathway leading to SUMOylation of Sp3. The free carboxyl group of the C-terminal glycine of SUMO forms an isopeptide bond with the ε-amino group of a lysine (K) in Sp3. The reaction is mediated by the ATP-dependent heterodimeric E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9 that form thioesters (S) with SUMO. (B) Affinity-purified epitope-tagged Sp3WT (lanes 1–3) and Sp3SD (lanes 5–7) were subjected to in vitro SUMOylation reactions in the presence or absence of recombinant E1, Ubc9 and SUMO-1 as indicated. Sp3 and SUMO-modified Sp3 (arrow) were detected by western blot analysis using anti-HA antibodies. Lane 4 (HA/FL-Sp3) contains whole-cell extract from Sp3-expressing SL2 cells. (C) Bacterially expressed GST fusion proteins GST–Sp3WT, GST–Sp3kee and GST–Sp3BID bound to GST–Sepharose were subjected to in vitro SUMOylation reactions in the presence or absence of recombinant E1, Ubc9 and SUMO-1 as indicated. The GST–Sp3BID protein contains the second glutamine-rich activation domain (B domain) and the ID with the IKEE motif lacking the transactivation domain A and the C-terminal DNA-binding domain of Sp3. In the GST–Sp3kee protein, the KEE wild-type sequence of the ID is replaced by three alanine residues. Reaction products were detected by western blot analysis using anti-Sp3 (αSp3) and anti-SUMO-1 (αSUMO-1) antibodies as indicated. Arrows point to the SUMOylated Sp3 fragments. (D) SUMO-1 and SUMO-2 were equally conjugated to Sp3. Epitope-tagged recombinant Sp3 wild-type (Sp3WT) or the Sp3SD mutant was subjected to SUMO modification with equal concentrations of SUMO-1 and SUMO-2 (5 ng/µl each). Detection was by immunoblotting with αHA antibodies. (E) DeSUMOylation of SUMO-1-modified Sp3 by the isopeptidase Ulp1. The GST–Sp3BID fragment (see panel C) bound to glutathione–Sepharose was SUMOylated in vitro and subsequently incubated with recombinant ULP1 isopeptidase at 16 or 30°C for 30 or 60 min, as indicated. Detection was by immunoblotting with αSp3 antibodies.

Article Snippet: The following antibodies and dilutions (in 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20, 1% skim milk) were used: mouse anti-GFP (Clontech) 1:2000; rabbit anti-Sp3 polyclonal IgG (Santa Cruz) 1:2000; rabbit anti-Sp3 serum ( Hagen et al ., 1994 ) 1:1000; rabbit anti-HA (Santa Cruz) 1:2000; rabbit anti-SUMO-1 (Santa Cruz) 1:1000; horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science) 1:10 000; and horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science) 1:10 000.

Techniques: In Vitro, Conjugation Assay, Affinity Purification, Recombinant, Modification, Western Blot, Expressing, Activation Assay, Binding Assay, Sequencing, Mutagenesis, Incubation

Journal:

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

doi: 10.1093/emboj/cdf510

Figure Lengend Snippet: Fig. 3. Identification of PIAS1 as an interaction partner of Sp3 and Ubc9. (A) Interaction of PIAS1 with the ID of Sp3 in Saccharomyces cerevisiae. Yeast cells containing a LexA-driven LacZ reporter construct were transformed with expression constructs for LexA, LexA-Sp3ID or LexA-Sp3ID/kee (baits) along with a construct in which the Gal4 activation domain is fused to the 500 C-terminal amino acids of PIAS1 (Gal4-PIAS1, prey). In the LexA-Sp3ID/kee construct, the KEE sequence of the SUMOylation motif is replaced by three alanine residues. β-galactosidase activity was visualized by addition of 0.5% X-gal to the agar. (B) In vitro association of PIAS1 with Sp3 and SUMO-1-modified Sp3. Sp3 (small isoform) was in vitro translated in the presence of [35S]methionine and subsequently subjected to in vitro SUMO-1 conjugation. The reaction that contained unmodified Sp3 and SUMO-modified Sp3 (lane 8) was incubated with similar amounts of the glutathione matrix (lane 2), immobilized GST (lane 3), GST–Ubc9 (lane 4) or GST–PIAS1 (lane 6). In lane 5, unmodified 35S-labelled Sp3 was incubated with GST–PIAS1. Bound Sp3 proteins were resolved by SDS–PAGE and visualized by fluorography. Lanes 7 and 8 contain 10% of the input 35S-labelled Sp3 protein. Numbers on the left indicate the molecular mass of protein markers in kDa. (C) In vitro association of Ubc9 with PIAS1. PIAS1 was in vitro translated in the presence of [35S]methionine and incubated with glutathione–Sepharose matrix (lane 2) or with ∼2 µg of immobilized GST (lanes 3 and 4) or GST–Ubc9 (lanes 5 and 6). Bound PIAS1 protein was resolved by SDS–PAGE and visualized by fluorography. Lane 7 contains 10% of the input 35S-labelled PIAS1 protein. Numbers on the left indicate the molecular mass of protein markers in kDa.

Article Snippet: The following antibodies and dilutions (in 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20, 1% skim milk) were used: mouse anti-GFP (Clontech) 1:2000; rabbit anti-Sp3 polyclonal IgG (Santa Cruz) 1:2000; rabbit anti-Sp3 serum ( Hagen et al ., 1994 ) 1:1000; rabbit anti-HA (Santa Cruz) 1:2000; rabbit anti-SUMO-1 (Santa Cruz) 1:1000; horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science) 1:10 000; and horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science) 1:10 000.

Techniques: Construct, Transformation Assay, Expressing, Activation Assay, Sequencing, Activity Assay, In Vitro, Modification, Conjugation Assay, Incubation, SDS Page

Journal:

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

doi: 10.1093/emboj/cdf510

Figure Lengend Snippet: Fig. 4. PIAS1 stimulates SUMO conjugation to Sp3. (A) Purified HA/FLAG-tagged Sp3 from SL2 cells was subjected to in vitro SUMO-1 (lanes 1–10) or SUMO-2 (lanes 11–16) modification in the presence of 10 mM glutathione (GSH; lanes 1 and 2), GST–PIAS1 (lanes 3–6 and 14–16) or GST (lanes 7–10 and 11–13). Reactions contained one-tenth of E1 and Ubc9 enzymes used in the experiments shown in Figure 2. After various time points, reactions were stopped by addition of Laemmli buffer. Proteins were resolved by SDS–PAGE and Sp3 detected by immunoblotting with αHA antibodies. (B) Schematic outline of the conjugation pathway leading to SUMO modification of Sp3. PIAS1 that interacted specifically with Ubc9 and SUMO-modified Sp3 acts as an E3 ligase for SUMO conjugation to Sp3.

Article Snippet: The following antibodies and dilutions (in 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20, 1% skim milk) were used: mouse anti-GFP (Clontech) 1:2000; rabbit anti-Sp3 polyclonal IgG (Santa Cruz) 1:2000; rabbit anti-Sp3 serum ( Hagen et al ., 1994 ) 1:1000; rabbit anti-HA (Santa Cruz) 1:2000; rabbit anti-SUMO-1 (Santa Cruz) 1:1000; horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science) 1:10 000; and horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science) 1:10 000.

Techniques: Conjugation Assay, Purification, In Vitro, Modification, SDS Page, Western Blot

Journal:

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

doi: 10.1093/emboj/cdf510

Figure Lengend Snippet: Fig. 6. Subcellular localization of Sp3 and SUMO-1 in MEFs and Ishikawa cells. (A) Sp3–/– MEFs were transfected with 1 µg of an expression construct for GFP–Sp3. The intracellular distribution of GFP–Sp3 was detected by intrinsic green fluorescence of the GFP tag. Endogenous SUMO-1 localization was detected with a rabbit anti-SUMO-1 antibody and a CY3-conjugated secondary antibody. (B) Ishikawa cells were transfected with 1 µg of an expression construct for GFP–SUMO-1. Visualization was by the intrinsic green fluorescence of the GFP moiety. Endogenous Sp3 localization was detected with a rabbit anti-Sp3 antibody and a CY3-conjugated secondary antibody.

Article Snippet: The following antibodies and dilutions (in 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20, 1% skim milk) were used: mouse anti-GFP (Clontech) 1:2000; rabbit anti-Sp3 polyclonal IgG (Santa Cruz) 1:2000; rabbit anti-Sp3 serum ( Hagen et al ., 1994 ) 1:1000; rabbit anti-HA (Santa Cruz) 1:2000; rabbit anti-SUMO-1 (Santa Cruz) 1:1000; horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science) 1:10 000; and horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science) 1:10 000.

Techniques: Transfection, Expressing, Construct, Fluorescence

Journal:

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

doi: 10.1093/emboj/cdf510

Figure Lengend Snippet: Fig. 7. SUMO-modified Sp3 binds specifically DNA. (A) A C-terminal Sp3 fragment (Sp3-320C) was subjected to in vitro conjugation with SUMO-1 in the absence (–) or presence (+) of enzymes. Subsequently, reactions were analysed by immunoblotting (top right, αSp3) and EMSA. All DNA-binding reactions contained 0.1 ng of 32P-labelled GC oligonucleotide and various amounts (1- to 20-fold molar excess) of unlabelled GC or HNF3 oligonucleotides, as indicated. (B) Purified HA/FLAG-tagged Sp3 was subjected to in vitro conjugation with SUMO-1 or SUMO-2 and analysed by immunoblotting (top right, αHA) and southwestern analysis. For southwestern analysis, SUMOylation reaction products were separated by 8% SDS–PAGE, transferred to nitrocellulose and subsequently incubated with 32P-labelled GC-box oligonucleotide in the absence (lanes 1– 3) or presence (lanes 4–6) of 100-fold molar excess of a specific competitor (GC box) or an unspecific (HNF3 site) (lanes 7–9) oligonucleotide. (C) Sp3 is not a target for SUMOylation when bound to DNA. Epitope-tagged recombinant Sp3 was subjected to SUMO-1 modification in the absence (lane 3) or presence of 10 and 100 ng of GC-box oligonucleotide (lanes 1 and 2) or HNF3-binding site oligonucleotide (lanes 4 and 5). Reaction products were separated by 8% SDS–PAGE and analysed by immunoblotting with αHA antibodies.

Article Snippet: The following antibodies and dilutions (in 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20, 1% skim milk) were used: mouse anti-GFP (Clontech) 1:2000; rabbit anti-Sp3 polyclonal IgG (Santa Cruz) 1:2000; rabbit anti-Sp3 serum ( Hagen et al ., 1994 ) 1:1000; rabbit anti-HA (Santa Cruz) 1:2000; rabbit anti-SUMO-1 (Santa Cruz) 1:1000; horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Life Science) 1:10 000; and horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Science) 1:10 000.

Techniques: Modification, In Vitro, Conjugation Assay, Western Blot, Binding Assay, Purification, SDS Page, Incubation, Recombinant

Journal:

Article Title: Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

doi: 10.1007/978-1-59745-566-4_11

Figure Lengend Snippet: (A) Time course for a single turnover assay for SUMO transfer from the E2-SUMO thioester to the C-terminal tetramerization domain of human p53 at various p53 concentrations after separation by SDS-PAGE and detection with an anti-SUMO-1 antibody. (B) Data in A depicted graphically to calculate the reaction rates at various p53 concentrations by linear regression analysis. (C) Reaction rates (Y-axis) plotted against various p53 concentrations (X-axis) and data fit to a rectangular hyperbola of the form y=ax/(b+x). The data points represent the mean of three independent experiments and error bars denote one standard deviation.

Article Snippet: Primary Antibody: Antibody against human SUMO-1 (Boston Biochem) used at 1:1000 dilution in blocking buffer.

Techniques: Turnover Assay, SDS Page, Standard Deviation

Journal:

Article Title: Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

doi: 10.1007/978-1-59745-566-4_11

Figure Lengend Snippet: (A) Time course for SUMO conjugation to p53 at 94 μM under single turnover conditions at various pH values at 4°C after separation by SDS-PAGE and detection with an anti-SUMO-1 antibody. (B) Data in A depicted graphically to calculate the reaction rates at various pH values. The reaction rates are calculated by linear regression analysis. (C) Initial reaction rates (Y-axis) plotted as a function of pH and data fit to a sigmoidal function (see text for details). The vertical lines mark the pH at half-maximal activity (pK of the titratable group). The data points represent the mean of two independent experimental trials and error bars denote one standard deviation.

Article Snippet: Primary Antibody: Antibody against human SUMO-1 (Boston Biochem) used at 1:1000 dilution in blocking buffer.

Techniques: Conjugation Assay, SDS Page, Activity Assay, Standard Deviation