Journal: eLife

Article Title: Sumoylation promotes optimal APC/C activation and timely anaphase

doi: 10.7554/eLife.29539

Figure Lengend Snippet: ( A ) HeLa cells were synchronized in S-phase using a double-thymidine arrest and released for varying time points. Whole cell lysates were analyzed by immunoblotting for APC4, Cyclin B1, Cdc20, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. Asterisks indicate sumoylated forms of APC4. ( B ) APC4 contains two C-terminal SUMO consensus site lysines at 772 and 798. ( C ) Full-length wild-type APC4 or the indicated lysine to alanine substitution mutants were expressed in rabbit reticulocyte lysate in the presence of [ 35 S]-methionine and incubated for the indicated times in modification reactions containing SUMO E1 and E2 enzymes and SUMO2. Proteins were detected by SDS-PAGE and autoradiography. Asterisks indicate sumoylated forms of APC4. ( D ) Constructs coding for FLAG-tagged versions of wild type APC4 or a sumoylation-deficient mutant containing arginine substitutions at lysines 772 and 798 (APC4 KR ) were used to generate stable inducible cell lines in YFP-H2B HeLa cells. ( E ) Endogenous APC4 was depleted by siRNA, and FLAG-APC4 or FLAG-APC4 KR stable cell lines were induced by doxycycline for 48 hr. Immunoblot analysis using APC4 and tubulin-specific antibodies reveals that FLAG-APC4 and FLAG-APC4 KR are expressed at near endogenous levels. ( F–G ) Co-immunoprecipitations were performed with an antibody against APC4, followed by immunoblotting for APC4 or SUMO2. FLAG-APC4 is sumoylated in vivo while FLAG-APC4 KR is not. Asterisks indicate sumoylated APC4.

Article Snippet: For immunoblotting, proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked in 5% milk in TS-T, and then probed with the following antibodies diluted in PBS supplemented with 2% BSA and 0.05% NaN 3 : APC2 (rabbit, a generous gift from Hongtao Yu), APC4 (rabbit, A301-176A, 1:2000, Bethyl Laboratories, Inc), BubR1 (rabbit, GeneTex, 1:1000) Cyclin B1 (mouse, GNS1: sc-245, 1:200, Santa Cruz Laboratories), Cdc20 (mouse, p55 CDC (E-7): sc-13–162, 1:250, Santa Cruz Laboratories), ECS (DDDDK, goat, A190-101A, Bethyl Laboratories, Inc.), GAPDH (rabbit, TAB1001, OpenBiosystems, 1:15,000), Myc (mouse, a generous gift from Hongtao Yu), SENP1 (rabbit, ab108981, 1:10,000, Abcam), SENP2 (rabbit polyclonal, produced as described previously: , 1:500), SUMO-1 (21C7, 1:100), SUMO-2/3 (8A2, 1:800), and Tubulin (mouse, DM1A, 1:5000, Sigma-Aldrich.

Techniques: Western Blot, Control, Incubation, Modification, SDS Page, Autoradiography, Construct, Mutagenesis, Stable Transfection, In Vivo

Journal: eLife

Article Title: Sumoylation promotes optimal APC/C activation and timely anaphase

doi: 10.7554/eLife.29539

Figure Lengend Snippet: ( A ) Cells were transfected with control or APC4-specific siRNAs for 48 hr followed by 16 hr of timelapse live cell acquisition. siAPC4 transfected cells were also induced to express FLAG-APC4 or FLAG-APC4 KR . Analysis represents mitotic progression time beginning with nuclear envelope breakdown (NEBD) to anaphase onset. Quantification of mitotic phenotypes is shown. Prolonged metaphase is defined by >60 min in metaphase plate alignment before anaphase onset. Abnormal metaphase is defined by inability to generate a metaphase plate and defects in chromosomal cohesion. n > 100 for each cell line. ( B ) Cells representative of each mitotic phenotype categorized in ( A ) are featured with timestamps in minutes. ( C ) Mitotic progression beginning with NEBD to metaphase plate alignment and from metaphase plate alignment to anaphase onset was quantified in FLAG-APC4 and FLAG-APC4 KR expressing cells. Experiments were performed in triplicate; means are displayed and error bars represent standard deviations. n = 50 for each cell line. Two-tailed t-tests were used to calculate significance: p=0.38 for differences in FLAG-APC4 and FLAG-APC4 KR timing from NEBD-metaphase, p<0.001 for differences in FLAG-APC4 and FLAG-APC4 KR timing from metaphase plate alignment to anaphase. ( D ) Individual timing of metaphase-anaphase progression is displayed for FLAG-APC4 and FLAG-APC4 KR expressing cells. n = 215 for each cell line. ( E ) Representative cells from timelapse acquisition beginning with NEBD to anaphase onset in FLAG-APC4 and FLAG-APC4 KR expressing cells with timestamps indicated in minutes. ( F ) FLAG-APC4 and ( G ) FLAG-APC4 KR expressing cells were synchronized in S-phase using a double-thymidine block and released for various time points. Whole cell lysates were analyzed by immunoblotting for APC4, Cyclin B1, Cdc20 and GAPDH as a loading control. Asterisks (*) indicate sumoylated forms of APC4. ( H ) Relative protein levels of Cyclin B1 in FLAG-APC4 and FLAG-APC4 KR cells were quantitated using ImageJ. Normalized mean values are graphed with standard deviations from three separate experiments.

Article Snippet: For immunoblotting, proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked in 5% milk in TS-T, and then probed with the following antibodies diluted in PBS supplemented with 2% BSA and 0.05% NaN 3 : APC2 (rabbit, a generous gift from Hongtao Yu), APC4 (rabbit, A301-176A, 1:2000, Bethyl Laboratories, Inc), BubR1 (rabbit, GeneTex, 1:1000) Cyclin B1 (mouse, GNS1: sc-245, 1:200, Santa Cruz Laboratories), Cdc20 (mouse, p55 CDC (E-7): sc-13–162, 1:250, Santa Cruz Laboratories), ECS (DDDDK, goat, A190-101A, Bethyl Laboratories, Inc.), GAPDH (rabbit, TAB1001, OpenBiosystems, 1:15,000), Myc (mouse, a generous gift from Hongtao Yu), SENP1 (rabbit, ab108981, 1:10,000, Abcam), SENP2 (rabbit polyclonal, produced as described previously: , 1:500), SUMO-1 (21C7, 1:100), SUMO-2/3 (8A2, 1:800), and Tubulin (mouse, DM1A, 1:5000, Sigma-Aldrich.

Techniques: Transfection, Control, Expressing, Two Tailed Test, Blocking Assay, Western Blot

Journal: eLife

Article Title: Sumoylation promotes optimal APC/C activation and timely anaphase

doi: 10.7554/eLife.29539

Figure Lengend Snippet: ( A ) Endogenous APC4 was depleted by siRNA, and FLAG-APC4 or FLAG-APC4 KR stable cell lines were induced by doxycycline for 48 hr. Immunoblot analysis using APC4 and tubulin-specific antibodies reveals that FLAG-APC4 and FLAG-APC4 KR are expressed at near endogenous levels. ( B ) Cells were treated with reversine immediately prior to 4 hr timelapse acquisition, and timing from NEBD to anaphase onset (mitotic exit timing) was collected. Data is representative of four independent experiments, n = 23 for each cell line. On average, FLAG-APC4 cells take 48.65 min to mitotic exit (Standard Deviation = SD = 15.99) while reversine treated FLAG-APC4 cells take 34.87 min (SD = 11.00). This difference is significant (p=0.0014). FLAG-APC4 KR cells take 81.48 min to mitotic exit (SD = 30.78) while reversine treated FLAG-APC4 KR cells take 78.78 min (SD = 21.41). This difference is not statistically significant (p=0.73). Two-tailed t-tests were used to calculate significance. ( C–H ) Representative cells from timelapse acquisition beginning with NEBD to anaphase onset in cells with or without 50 nM or 1 μM reversine are shown with timestamps indicated in minutes. ( I ) Activity assays using unmodified or in vitro sumoylated APC/C was performed with recombinant myc-Securin substrate, in the presence or absence of recombinant co-activator Cdc20. ( J–K ) Immunoblots of immunopurified APC/C using antibodies specific for APC4 and SUMO2/3 with SUMO modification of APC/C complex indicated with ‘*”. ( L ) Activity assays using unmodified or in vitro sumoylated APC/C was performed with recombinant myc-Securin substrate, in the presence or absence of recombinant co-activator Cdc20 and inhibitory MCC complex as indicated. ( M–O ) Immunoblots of immunopurified APC/C using antibodies specific for APC4, APC3 and SUMO2/3, with SUMO modification of APC/C complex indicated with ‘*”.

Article Snippet: For immunoblotting, proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked in 5% milk in TS-T, and then probed with the following antibodies diluted in PBS supplemented with 2% BSA and 0.05% NaN 3 : APC2 (rabbit, a generous gift from Hongtao Yu), APC4 (rabbit, A301-176A, 1:2000, Bethyl Laboratories, Inc), BubR1 (rabbit, GeneTex, 1:1000) Cyclin B1 (mouse, GNS1: sc-245, 1:200, Santa Cruz Laboratories), Cdc20 (mouse, p55 CDC (E-7): sc-13–162, 1:250, Santa Cruz Laboratories), ECS (DDDDK, goat, A190-101A, Bethyl Laboratories, Inc.), GAPDH (rabbit, TAB1001, OpenBiosystems, 1:15,000), Myc (mouse, a generous gift from Hongtao Yu), SENP1 (rabbit, ab108981, 1:10,000, Abcam), SENP2 (rabbit polyclonal, produced as described previously: , 1:500), SUMO-1 (21C7, 1:100), SUMO-2/3 (8A2, 1:800), and Tubulin (mouse, DM1A, 1:5000, Sigma-Aldrich.

Techniques: Stable Transfection, Western Blot, Standard Deviation, Two Tailed Test, Activity Assay, In Vitro, Recombinant, Modification

Journal: eLife

Article Title: Sumoylation promotes optimal APC/C activation and timely anaphase

doi: 10.7554/eLife.29539

Figure Lengend Snippet: Stable inducible cell lines were depleted of endogenous APC4 using siRNA for 48 hr with concomitant induction of FLAG-APC4 or FLAG-APC4 KR expression and stained with antibodies specific to ( A ) Mad1, ( B ) Mad2, ( C ) BubR1, and ( D ) Cdc20. Chromatin (colored in teal) was visualized by detection of YFP-H2B expressed in these cell lines. ( E and F ) Inducible cell lines were treated as described above. Cells were stained with FLAG and CREST-specific antibodies and analyzed by immunofluorescence microscopy. Boxed regions are magnified to show co-localization at kinetochores during prometaphase and metaphase.

Article Snippet: For immunoblotting, proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, blocked in 5% milk in TS-T, and then probed with the following antibodies diluted in PBS supplemented with 2% BSA and 0.05% NaN 3 : APC2 (rabbit, a generous gift from Hongtao Yu), APC4 (rabbit, A301-176A, 1:2000, Bethyl Laboratories, Inc), BubR1 (rabbit, GeneTex, 1:1000) Cyclin B1 (mouse, GNS1: sc-245, 1:200, Santa Cruz Laboratories), Cdc20 (mouse, p55 CDC (E-7): sc-13–162, 1:250, Santa Cruz Laboratories), ECS (DDDDK, goat, A190-101A, Bethyl Laboratories, Inc.), GAPDH (rabbit, TAB1001, OpenBiosystems, 1:15,000), Myc (mouse, a generous gift from Hongtao Yu), SENP1 (rabbit, ab108981, 1:10,000, Abcam), SENP2 (rabbit polyclonal, produced as described previously: , 1:500), SUMO-1 (21C7, 1:100), SUMO-2/3 (8A2, 1:800), and Tubulin (mouse, DM1A, 1:5000, Sigma-Aldrich.

Techniques: Expressing, Staining, Immunofluorescence, Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: Schwann Cell Expressed Nogo-B Modulates Axonal Branching of Adult Sensory Neurons Through the Nogo-B Receptor NgBR

doi: 10.3389/fncel.2015.00454

Figure Lengend Snippet: Immunohistochemistry of Nogo-B in the sciatic nerve. (A) The two isoforms Nogo-A and Nogo-B differ considerably in size and function. A common feature is the C-terminal RHD which harbors not only an ER anchoring motif at the very C-terminus but also two hydrophobic sequence stretches (shaded in gray); they are believed to contribute to ER tubulation by inducing membrane curvature. Nogo-A is selectively recognized by the monoclonal 11C7 antibody while the epitope of the Bianca antiserum resides within the NIR domain that is shared by both isoforms. The NEP1–40 peptide is derived from and blocks the signaling activities of the Nogo-66 loop domain. The Nogo-B specific receptor NgBR, and recombinant NgBR-Ecto, bind to the AmNogo-B sequence stretch that is composed of parts of the NIR domain and RHD. (B) Immunolabeling of cross sections of the sciatic nerve of a wildtype and nogo-a/b knockout mouse. The external cytoplasmic compartment of Schwann cells is immunoreactive for Nogo. The myelin sheath that is visualized with myelin basic protein (MBP) is devoid of Nogo. Axons are revealed with a neurofilament antibody. Single confocal sections are shown. Scale bar is 10 μm. (C) Immunolabeling of cross sections of the rat sciatic nerve with the Bianca antiserum and counter-staining with 11C7. Schwann cells are strongly positive for Bianca but not for 11C7 indicating that the majority of Bianca immunoreactivity is due to Nogo-B. In contrast, the Bianca immunoreactivity in axons seems to be due to Nogo-A as demonstrated by the 11C7 signal (white arrows). Scale bar is 5 μm. A single confocal section is shown. (D) Immunolabeling of teased nerve fibers of the sciatic nerve of an adult wildtype mouse. There is prominent Nogo immunoreactivity in Cajal bands and, to a lesser extent, in Schmidt-Lanterman incisures (arrow heads) and transverse trabeculae (triangles) as revealed by MAG and Vimentin co-distribution. The Nogo staining typically extends into nodes of Ranvier (arrows). Single confocal sections are shown. Scale bar is 10 μm.

Article Snippet: For detection the following primary antibodies were used: rabbit Bianca antiserum (1:20,000), rabbit NgBR antibody (1:400; proteintech #15986-1-AP), rabbit PO antibody (1:500; proteintech #10572-1-AP), rabbit p75 NTR antibody (1:1000; Promega #G3231), mouse actin antibody (1:10,000; Chemicon #MAB1501), mouse L-caldesmon (1:500; BD Biosciences #610660).

Techniques: Immunohistochemistry, Sequencing, Membrane, Derivative Assay, Recombinant, Immunolabeling, Knock-Out, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Schwann Cell Expressed Nogo-B Modulates Axonal Branching of Adult Sensory Neurons Through the Nogo-B Receptor NgBR

doi: 10.3389/fncel.2015.00454

Figure Lengend Snippet: Blocking the Nogo-B receptor NgBR mimics the loss-of- nogo branching phenotype. (A) Quantification of morphological parameters of adult wildtype sensory neurons grown on wildtype Schwann cells in the presence or absence of NEP1–40. Five independent experiments were carried out with a total of 181 neurons being analyzed in the control and 177 neurons in the NEP1–40 group. Morphological parameters were normalized to the control (PBS; set to 1.0) and expressed as mean ± SEM. P -values ≤ 0.05 are indicated. Neuronal cell body diameters are depicted in a dot blot. (B) Immunocytochemistry of a Schwann cell neuron co-culture stained for NgBR, Hoechst and neurofilament. Scale bar is 10 μm. (C) Co-immunoprecipitation experiments with a co-culture of wildtype Schwann cells and nogo-a/b deficient sensory neurons. For pull-down Bianca antiserum or a rabbit control IgG was used. Detection was done with Bianca antiserum and an NgBR antibody. Asterisks indicate the rabbit IgG heavy chain that is detected by the secondary anti-rabbit antibody. (D) Quantification of morphological parameters of adult wildtype sensory neurons grown on wildtype Schwann cells in the presence or absence of NgBR-Ecto. Five independent experiments were carried out with a total of 142 neurons being analyzed in the control and 167 neurons in the NgBR-Ecto group. Morphological parameters were normalized to the control (50% glycerol in PBS; set to 1.0) and expressed as mean ± SEM. P -values ≤ 0.05 are indicated. In the presence of NgBR-Ecto TNIS is reduced to 0.75 and TNL to 0.74 relative to the control levels. Neuronal cell body diameters are depicted in a dot blot.

Article Snippet: For detection the following primary antibodies were used: rabbit Bianca antiserum (1:20,000), rabbit NgBR antibody (1:400; proteintech #15986-1-AP), rabbit PO antibody (1:500; proteintech #10572-1-AP), rabbit p75 NTR antibody (1:1000; Promega #G3231), mouse actin antibody (1:10,000; Chemicon #MAB1501), mouse L-caldesmon (1:500; BD Biosciences #610660).

Techniques: Blocking Assay, Control, Dot Blot, Immunocytochemistry, Co-Culture Assay, Staining, Immunoprecipitation