mouse anti ranbp2  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Sumoylation of Human Argonaute 2 at Lysine-402 Regulates Its Stability

doi: 10.1371/journal.pone.0102957

Figure Lengend Snippet: ( A ) Ago2 is modified by SUMO1 and SUMO2 in vitro . 35 S-labelled in vitro- translated Ago2 was incubated in a sumoylation mix containing purified SAE1–SAE2, Ubc9 and SUMO1 or SUMO2, in the absence (-) or presence (+) of ATP. Reaction products were visualized by SDS–PAGE and autoradiography. ( B ) Sumoylation of Ago2 in vivo . HeLa cells were transfected with expression vectors for HA-tagged Ago2, SUMO1, His-SUMO1, SUMO2 and His-SUMO2 as indicated and analyzed by Western blot by anti-Ago2 antibody. ( C ) Ubc9 interacts with Ago2 in vivo . HeLa cells were transfected with expression vectors for HA-Ago2 and Ubc9 as indicated. Cell lysates were immunoprecipitated (IP) with mouse anti-HA antibody or control antibody (IgG) and probed with anti-HA and anti-Ubc9 antibodies. WCE, whole-cell extract, 2% of amount used in IP. ( D ) SUMO E3 ligase RanBP2 enhances sumoylation of Ago2 in vitro . In vitro sumoylation of 35 S-labelled, in vitro -translated human Ago2 in the absence or presence of RanBP2 or GST control. Reactions were incubated for indicated times (t = 0 min or t = 60 min) with 10 ng (1x) or 3 ng (0.3x) recombinant Ubc9. Reaction products were visualized by SDS–PAGE and autoradiography. Each blot is a representative of three independent experiments.

Article Snippet: For western blot, the following antibodies were used: rabbit anti-Ago2 (Sigma) at 1∶1000, mouse anti-HA (16B12, Covance) at 1∶2000, mouse anti-Ubc9 (BD biosciences) at 1∶1000, mouse anti-tubulin (Sigma) at 1∶10 000, in house rabbit anti-SUMO1 at 1∶1000, mouse anti-RanBP2 (Santa Cruz) at 1∶400, rabbit anti-SAE2 (Abcam) at 1∶1000, mouse anti-Ago1 (Upstate) at 1∶1000, mouse anti-Vinculin (Abcam) at 1/1000, rabbit anti-PARP1 (Santa Cruz) at 1∶1000 and mouse anti-GFP (Sigma) at 1∶500.

Techniques: Modification, In Vitro, Incubation, Purification, SDS Page, Autoradiography, In Vivo, Transfection, Expressing, Western Blot, Immunoprecipitation, Recombinant

Journal: PLoS ONE

Article Title: Sumoylation of Human Argonaute 2 at Lysine-402 Regulates Its Stability

doi: 10.1371/journal.pone.0102957

Figure Lengend Snippet: ( A ) Sumoylated Ago2 resides both in the cytosol and nucleus. HeLa cells were transfected with the indicated plasmids and fractionated to analyze nuclear and cytoplasmic proteins. HA-Ago2 was detected by anti-HA antibody. Immunoblots for endogenous PARP1, a nuclear protein, and endogenous vinculin, a cytoplasmic protein, were performed to validate nuclear-cytoplasmic fractionation. ( B ) Sumoylation is dispensable for subcellular localization of Ago2. HeLa cells were transfected with wild type or sumoylation mutants (4KR and K402R) of HA-Ago2, and treated as indicated (250 µM sodium arsenite -NaAsO 2 - for 60 min). Ago2 localization was determined by immunofluorescence using the anti-HA antibody (red). The cells were also immunostained for Dcp2 (marking P-bodies in green). The nuclei are stained with DAPI (blue). Ago2 staining was diffuse in the cytoplasm, as well as concentrated in cytoplasmic P-bodies (marked by arrows in untreated cells). NaAsO 2 treatment increases size and number of P-bodies. ( C ) Endogenous Ago2 (red) and RanBP2 (green) colocalize in the nuclei of HeLa cells (indicated by arrowheads).

Article Snippet: For western blot, the following antibodies were used: rabbit anti-Ago2 (Sigma) at 1∶1000, mouse anti-HA (16B12, Covance) at 1∶2000, mouse anti-Ubc9 (BD biosciences) at 1∶1000, mouse anti-tubulin (Sigma) at 1∶10 000, in house rabbit anti-SUMO1 at 1∶1000, mouse anti-RanBP2 (Santa Cruz) at 1∶400, rabbit anti-SAE2 (Abcam) at 1∶1000, mouse anti-Ago1 (Upstate) at 1∶1000, mouse anti-Vinculin (Abcam) at 1/1000, rabbit anti-PARP1 (Santa Cruz) at 1∶1000 and mouse anti-GFP (Sigma) at 1∶500.

Techniques: Transfection, Western Blot, Fractionation, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Sumoylation of Human Argonaute 2 at Lysine-402 Regulates Its Stability

doi: 10.1371/journal.pone.0102957

Figure Lengend Snippet: ( A ) Depletion of SUMO enzymes results in increased steady-state Ago2 protein levels. HeLa cells were transfected with control siRNA (scr) or siRNA against Ubc9, SAE2 or RanBP2. Relative levels of Ago2 are indicated in the graph. Means of three independent experiments. Significant p-values are shown. ( B ) Loss of Ubc9 in vivo results in enhanced endogenous Ago2 protein levels. Ubc9 +/+ /ROSA26-CreERT2 control (Ubc9 +/+ ) and Ubc9 fl/− /ROSA26-CreERT2 conditional knockout (Ubc9 −/− ) adult mice (n = 3, #1, #2, #3) received 3 consecutive days of 4-OHT injection. Proteins were extracted from heart, liver and skeletal muscle and were analyzed by Western blot. Quantification of Ago2 protein levels in organs after normalization to the ponceau staining is shown. Bars represent means and standard deviations. from 3 mice (white bars: Ubc9 +/+ , black bars: Ubc9 −/− ). P-values, determined by two-tailed Student’s t-test, are indicated.

Article Snippet: For western blot, the following antibodies were used: rabbit anti-Ago2 (Sigma) at 1∶1000, mouse anti-HA (16B12, Covance) at 1∶2000, mouse anti-Ubc9 (BD biosciences) at 1∶1000, mouse anti-tubulin (Sigma) at 1∶10 000, in house rabbit anti-SUMO1 at 1∶1000, mouse anti-RanBP2 (Santa Cruz) at 1∶400, rabbit anti-SAE2 (Abcam) at 1∶1000, mouse anti-Ago1 (Upstate) at 1∶1000, mouse anti-Vinculin (Abcam) at 1/1000, rabbit anti-PARP1 (Santa Cruz) at 1∶1000 and mouse anti-GFP (Sigma) at 1∶500.

Techniques: Transfection, In Vivo, Knock-Out, Injection, Western Blot, Staining, Two Tailed Test

Journal: eLife

Article Title: T-cell receptor (TCR) signaling promotes the assembly of RanBP2/RanGAP1-SUMO1/Ubc9 nuclear pore subcomplex via PKC-θ-mediated phosphorylation of RanGAP1

doi: 10.7554/eLife.67123

Figure Lengend Snippet: ( A, B ) Reciprocal IP analysis of the association between HA-tagged wild-type or K524-mutated RanGAP1 and endogenous Ubc9. RanGAP1 expression was analyzed by anti-HA antibody immunoblotting in WCL (bottom panels). RanGAP1 AA (S504A/S506A); RanGAP1 EE (S504E/S506E). ( C–E ) Immunoblot analysis of HA-RanGAP1 IPs ( C ) and Ubc9 IPs or RanBP2 IPs ( D ) from Jurkat-TAg cells transfected with HA-RanGAP1 and HA-RanGAP1 mutants. The ratio of RanGAP1-SUMO1 to RanGAP1 in the WCL of ( C ) and ( D ) is quantified in ( E ); quantification is based on three biological replicates. *p<0.05 (one-way ANOVA with post hoc test). Data are representative of three biological replicates. Figure 5—source data 1. Uncropped western blot for . Figure 5—source data 2. Row data for .

Article Snippet: Antibody , Mouse monoclonal anti- RanBP2 , Santa Cruz Biotechnology , Cat #: sc-74518, RRID: AB_2176784 , WB (1:1000).

Techniques: Expressing, Western Blot, Transfection

Journal: eLife

Article Title: T-cell receptor (TCR) signaling promotes the assembly of RanBP2/RanGAP1-SUMO1/Ubc9 nuclear pore subcomplex via PKC-θ-mediated phosphorylation of RanGAP1

doi: 10.7554/eLife.67123

Figure Lengend Snippet: Upon TCR stimulation, PKC-θ phosphorylates RanGAP1 to increase its association with Ubc9, thereby enhancing the sumoylation of RanGAP1, which is required for assembly of the RanBP2/RanGAP1-SUMO1/Ubc9 subcomplex. This complex then promotes the nuclear import of NF-ATc1, NF-κB, and AP-1.

Article Snippet: Antibody , Mouse monoclonal anti- RanBP2 , Santa Cruz Biotechnology , Cat #: sc-74518, RRID: AB_2176784 , WB (1:1000).

Techniques:

Journal: eLife

Article Title: T-cell receptor (TCR) signaling promotes the assembly of RanBP2/RanGAP1-SUMO1/Ubc9 nuclear pore subcomplex via PKC-θ-mediated phosphorylation of RanGAP1

doi: 10.7554/eLife.67123

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti- RanBP2 , Santa Cruz Biotechnology , Cat #: sc-74518, RRID: AB_2176784 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Retroviral, Sequencing, CRISPR, Software