monoclonal mouse antibody against sumo1  (Santa Cruz Biotechnology)

Journal: Journal of Molecular Cell Biology

Article Title: SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

doi: 10.1093/jmcb/mjaa076

Figure Lengend Snippet: α-tubulin is SUMOylated in cells and in vitro . ( A ) Immunoprecipitates with α-tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were subject to immunoblotting (IB) and probed with Flag and α-tubulin (α-Tub) Abs. ( B ) Endogenous α-tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. ( C ) Immunoprecipitates with α-tubulin Ab from HEK293 cells expressing HA-Ubc9 were probed with HA and α-tubulin Abs. ( D ) Coomassie blue staining of purified mouse brain tubulin including α-tubulin and β-tubulin, indicated by arrows. ( E ) Coomassie blue staining of purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and His-SUMO1ΔGG. Asterisk indicates the band of purified protein. ( F ) In vitro SUMOylation assay using purified GST-SAE2/1, GST-Ubc9, His-SUMO1GG, and brain tubulins. ( G ) Ratio of density of SUMOylated bands to unSUMOylated bands. ( H ) Purified tubulin was in vitro SUMOylated and probed with α-tubulin Ab. ( I ) In vitro SUMOylation assay using soluble tubulins and MTs. ( J ) Immunoprecipitates with β-tubulin Ab from HEK293 cells expressing Flag-SUMO1, Flag-SUMO2, or Flag-SUMO3 were probed with Flag and β-tubulin Abs. ( K ) Purified tubulin was in vitro SUMOylated and probed with β-tubulin Ab. ( L ) Endogenous β-tubulin in HEK293 cells was immunoprecipitated and probed with SUMO1 Ab. The experiments were repeated three times.

Article Snippet: The primary antibodies included mouse antibodies against SUMO1 (1:1000; CST, 4940S), SUMO2/3 (1:1000; CST, 4971S), α-tubulin (1:10000; Sigma, T9026), α-tubulin (1:10000; Abcam, ab18251), Flag (1:10000; Sigma, F7425), β-actin (1:50000; Chemicon, MAB1501), and GAPDH (1:10000; Abcam, ab8245).

Techniques: In Vitro, Expressing, Western Blot, Immunoprecipitation, Staining, Purification

Journal: Journal of Molecular Cell Biology

Article Title: SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

doi: 10.1093/jmcb/mjaa076

Figure Lengend Snippet: SUMOylation is mainly enriched in soluble α-tubulin. ( A ) PLA with α-tubulin and SUMO1 Abs was performed in HEK293 cells. Confocal images of PLA signals and tubulin labelled after PLA are shown. The enlarged image of the boxed area is shown at the lower right. Scale bar, 10 μm. ( B ) PLA dots on and off MTs were quantified. n = 11 cells. ( C ) Soluble and polymerized tubulins were separated in SUMO1-overexpressing HEK293 cells and subject to IP using α-tubulin Ab. Immunoprecipitates were detected by SUMO1 Ab. The experiments were repeated three times. ( D ) Quantification of the SUMOylation in soluble or polymerized tubulins. Data are mean ± SEM from three independent experiments.

Article Snippet: The primary antibodies included mouse antibodies against SUMO1 (1:1000; CST, 4940S), SUMO2/3 (1:1000; CST, 4971S), α-tubulin (1:10000; Sigma, T9026), α-tubulin (1:10000; Abcam, ab18251), Flag (1:10000; Sigma, F7425), β-actin (1:50000; Chemicon, MAB1501), and GAPDH (1:10000; Abcam, ab8245).

Techniques:

Journal: Journal of Molecular Cell Biology

Article Title: SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

doi: 10.1093/jmcb/mjaa076

Figure Lengend Snippet: α-tubulin is SUMOylated at K96, K166, and K304 and deSUMOylated by SENP1. ( A ) Alignment of lysines (in red color) and surrounding sequences of α-tubulin isotypes in the mouse. ( B ) Immunoprecipitates with Flag M2 beads from HEK293T cells expressing HA-SUMO1 with Flag-tagged isotypes of α-tubulin as indicated were probed with SUMO1 and Flag Abs. ( C ) Flag-tagged wild-type (WT) and various α1A mutant at MS-predicted SUMOylation sites were expressed with HA-SUMO1 in CHO-K1 cells and then immunoprecipitated. Immunoprecipitates were detected by SUMO1 and Flag Abs. ( D ) List of putative SUMOylation sites on α1A isotype predicted by different softwares. ( E ) Flag-tagged WT and combined mutant α1A at bioinformatics-predicted SUMOylation sites were expressed with HA-SUMO1 in CHO-K1 cells and then immunoprecipitated. Immunoprecipitates were detected by SUMO1 and Flag Abs. ( F ) Schematic representation of SUMOylation sites on the structure of α-tubulin. K96, K166, and K304 are shown as red spheres. ( G ) Location of SUMOylation sites and the interface for lateral contact between two α-tubulins in one MT. K96, K166, and K304 are shown as red spheres, and H2-S3 loop and M-loop are in blue. ( H ) Flag-α1A was overexpressed with HA-SUMO1 or HA-SUMO1-K7,16,17R mutant in HEK293 cells. Immunoprecipitates using Flag M2 beads were probed with HA and Flag Abs. ( I ) Immunoprecipitates with Flag M2 beads from HEK293 cells expressing Flag-SENP1 or Flag-SENP2 were probed with Flag and α-tubulin Abs. ( J ) Immunoprecipitates with α-tubulin from HEK293 cells expressing Flag-SENP1 or Flag-SENP2 with Flag-SUMO1 were probed with SUMO1 and α-tubulin Abs. ( K ) Immunoprecipitates from E13.5 brain of SENP1 +/+ and SENP1 –/– mice were probed with SUMO1 and α-tubulin Abs. The experiments were repeated three times.

Article Snippet: The primary antibodies included mouse antibodies against SUMO1 (1:1000; CST, 4940S), SUMO2/3 (1:1000; CST, 4971S), α-tubulin (1:10000; Sigma, T9026), α-tubulin (1:10000; Abcam, ab18251), Flag (1:10000; Sigma, F7425), β-actin (1:50000; Chemicon, MAB1501), and GAPDH (1:10000; Abcam, ab8245).

Techniques: Expressing, Mutagenesis, Immunoprecipitation

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: HCN2 is SUMOylated in rat DRG. Denaturing immunoprecipitation (IP) experiments were performed on a DRG membrane preparation with an antibody against HCN2 or IgG (control). IP products from 1 membrane preparation were run in triplicate on an SDS‐polyacrylamide gel followed by western blotting (WB). The blot was cut into 3, and probed for HCN2, SUMO2/3 and SUMO1. The experiment was repeated using three different DRG membrane preparations. A representative result from one experiment is shown. All three WB antibodies recognized the same ~100 kD protein in the HCN2 IP product but not the IgG IP product. The data indicate that SUMO1 and SUMO2/3 are covalently linked to HCN2 since they remained bound under denaturing conditions. The ~50 kD band present in all lanes represents the IP antibody. DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Immunoprecipitation, Western Blot

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: Measuring HCN2 channel SUMOylation. (a) Verification of SUMO antibodies. Antibodies were (right) or were not (left) preabsorbed with the corresponding peptide for SUMO2/3 (upper panel) or SUMO1 (lower panel). SUMO is predominately expressed in nuclei. Note the loss of nuclear staining following preabsorption, for example, white arrows in bottom panel. Scale bars are 100 µm. (b) Method for quantifying HCN2 channel SUMOylation. In situ PLA was performed on DRG cryosections with (experimental) or without (control) antibodies against SUMO2/3 and HCN2. Upper panel shows a 5 µm projection of confocal optical slices through representative cells from control and experimental treatment groups. Each image represents a projection of five slices in continuous succession that together encompass the centre of the cell. Each optical section is 0.9 µm with a z‐stack interval of 1 µm. Note that the red puncta indicating SUMOylated HCN2 channels were largely absent when antibodies were omitted. Scale bar is 10 µm. The cells were circle and thresholded, and the resulting image is shown in the lower panel. Black puncta within the circled region were counted using the analyse particle tool on imageJ and normalized by the area (µm 2 ) of the circle. Note that all black puncta, regardless of size were counted. (c) HCN2 channels are SUMOylated in DRG neurons. Plots of puncta per µm 2 show there are significantly more puncta when primary antibodies for HCN2 and SUMO2/3 were included (0.08 ± 0.01 vs. 0.16 ± 0.02, p = 0.0149, paired t ‐test, n = 3, 70 and 62 cells analysed in total) *, p < 0.05. The lines indicate that the cells were from the same experiment, that is, alternate sections from a single DRG on the same slide receiving the same PLA reagents and treated in an identical fashion. DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Staining, In Situ, Ligation

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: HCN2 channel SUMOylation by SUMO1 is diminished in medium and large diameter neurons from ipsilateral relative to contralateral DRG at 1 day post‐CFA. (a) Representative PLA. Scale bars are 25 µm. Note that this image provides an overview, but for the purpose of quantification, individual projections were made for each cell, for example, Figure . (b) Plots of mean number of puncta/µm 2 for all DRG from six CFA treated animals on day 1 post‐CFA The number inside the bar represents the total number of cells analysed. Inset: Each line compares means for contralateral and ipsilateral DRG from one animal. Paired t ‐tests indicate medium and large diameter cells contain significantly fewer puncta in the ipsilateral DRG relative to the contralateral DRG (small, 0.33 ± 0.05 vs. 0.27 ± 0.03, p = 0.2058; medium, 0.34 ± 0.05 vs. 0.24 ± 0.03, p = 0.0480; large, 0.37 ± 0.05 vs. 0.26 ± 0.02, p = 0.0238), * p < 0.05. (c) Plots of mean puncta intensity on day 1 post‐CFA. Paired t ‐tests indicate mean puncta intensities were not significantly different for any size category in ipsilateral compared to contralateral DRG (small, 44.68 ± 4.03 vs. 41.48 ± 2.66, p = 0.5986; medium, 42.7 ± 3.24 vs. 45.65 ± 4.06, p = 0.6299; large, 44.44 ± 3.68 vs. 46.39 ± 4.27, p = 0.7631). CFA, Complete Freund's Adjuvant; DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Ligation

Journal: European Journal of Pain (London, England)

Article Title: Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

doi: 10.1002/ejp.1606

Figure Lengend Snippet: HCN2 channel SUMOylation by SUMO1 is increased in small neurons from ipsilateral relative to contralateral DRG at 3 days post‐CFA. (a) Representative PLA. Scale bars are 25 µm. Note that this image provides an overview, but for the purpose of quantification, individual projections were made for each cell, for example, Figure . (b) Plots of mean puncta/µm 2 for all DRG from six CFA treated animals on day 3 post‐CFA. The number inside the bar represents the total number of cells analysed. Inset: Each line compares means for contralateral and ipsilateral DRG from one animal. Significantly more puncta were observed in ipsilateral relative to contralateral DRG as indicated by paired t ‐tests or non‐parametric alternative** (small, 0.25 ± 0.03 vs. 0.32 ± 0.03, p = 0.0285; medium, 0.29 ± 0.05 vs. 0.25 ± 0.06, p = 0.4310; large**, 0.32 ± 0.03 vs. 0.28 ± 0.06, p = 0.5625). *, p < 0.05 (c) Plots of mean puncta intensity on day 3 post‐CFA. Paired t ‐tests indicate mean puncta intensities were not significantly different for any size category in ipsilateral compared to contralateral DRG (small, 46.14 ± 3.0 vs. 46.08 ± 1.41, p = 0.9826; medium, 44.58 ± 4.12 vs. 46.45 ± 1.32, p = 0.6612; large, 44.99 ± 3.38 vs. 48.35 ± 1.27 p = 0.2758). CFA, Complete Freund's Adjuvant; DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, s mall u biquitin like mo difier

Article Snippet: For PLA, a monoclonal mouse antibody against SUMO1 (Santa Cruz, Sc‐5308) was used at 1:100, and a monoclonal mouse antibody against SUMO2/3 (Developmental Studies Hybridoma Bank, SUMO‐2 8A2) was used at a concentration of 1:70 and developed by Matinus, M. at John Hopkins School of Medicine, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Ligation