rabbit polyclonal anti-sumo1 antibody fl-101  (Santa Cruz Biotechnology)

Journal: Journal of Virology

Article Title: SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms

doi: 10.1128/JVI.00871-20

Figure Lengend Snippet: AAV2 capsids are SUMOylated. Shown is an analysis of purified AAV2 particles. To determine AAV2 capsid modification by SUMO, we analyzed AAV2 vectors after purification by iodixanol gradient centrifugation. (a and b) AAV2 capsids and AAV1, -5, -8, and -9 capsids produced in 293T cells and analyzed by anti-SUMO1-specific antibodies or an antibody specific for all three AAV capsid proteins (B1 antibody). (c) Strategy to produce non-SUMOylated (n-s-) AAV2. HEK293T-Gam1 cells were transfected with three plasmids for AAV vector production. Induction of the avian adenovirus CELO Gam1 protein by doxycycline (Dox) leads to inactivation of the Sae1/2 E1 complex, leading to inactivation of the SUMOylation machinery. (d) Western blot analysis of Gam1 protein expression over time after Dox induction using Myc-tag antibody. HeLa-Gam1 cells were induced with 50 ng/ml Dox, and then cell lysate was harvested after 1, 3, 6, 9, 12, and 24 h. (e) Induction of Gam1 leads to higher transduction rates by AAV. HEK293T-Gam1 cells were incubated with and without Dox and then transduced with (SUMOylated) AAV-Luc vectors. RLU, relative light units. (f) Western blot analysis of AAV2 particles produced in 293T-Gam1 cells with (n-s-AAV2) or without Dox induction using SUMO1 and VP-specific antibodies. (g) Immunoprecipitation of AAV capsids. Purified AAV2 capsids were precipitated either with the AAV capsid-specific antibody A20 or with an anti-SUMO1 antibody. Precipitated proteins were then detected with either VP-specific antibody B1 or anti-SUMO1 antibody. As a specificity control, purified human papillomavirus type 58 capsids were precipitated using an unrelated antibody (K18L2).

Article Snippet: The membrane was blocked in 5% milk PBS-T at RT and then incubated with the desired primary antibody against SUMO1 (1:500; FL-101; Santa Cruz), SUMO2/3 (PA5-11373; 1:1,000; Sigma), DAXX (25c12, 1:1,000; CST), HA (1:500; sc-805; Santa Cruz), α-actin (1:5,000; MP), and VP (1:500 B1; Progen) in 5% PBS-T milk overnight at 4°C.

Techniques: Purification, Modification, Gradient Centrifugation, Produced, Transfection, Plasmid Preparation, Western Blot, Expressing, Transduction, Incubation, Immunoprecipitation, Control

Journal: Journal of Virology

Article Title: SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms

doi: 10.1128/JVI.00871-20

Figure Lengend Snippet: VP2 is a target for SUMOylation. (a) AAV2 vectors lacking either VP2, VP1, or VP1/2 were produced and purified by iodixanol gradient centrifugation. Vectors were analyzed by Western blotting using an anti-SUMO1 or the VP-specific antibody B1. (b) Analysis of AAV2 vectors carrying an N-terminally modified VP2. Vectors were produced as described for panel a. Lane 4 shows AAV vectors carrying a VP2 N-terminally fused GFP, and in lane 5 the HA tag is fused to the N terminus of VP2. (c) Detection of VP using a polyclonal antibody.

Article Snippet: The membrane was blocked in 5% milk PBS-T at RT and then incubated with the desired primary antibody against SUMO1 (1:500; FL-101; Santa Cruz), SUMO2/3 (PA5-11373; 1:1,000; Sigma), DAXX (25c12, 1:1,000; CST), HA (1:500; sc-805; Santa Cruz), α-actin (1:5,000; MP), and VP (1:500 B1; Progen) in 5% PBS-T milk overnight at 4°C.

Techniques: Produced, Purification, Gradient Centrifugation, Western Blot, Modification

Journal: Journal of Virology

Article Title: SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms

doi: 10.1128/JVI.00871-20

Figure Lengend Snippet: Lysine residues located in the N terminus of VP2 are essential for SUMOylation. (a) Sequence of VP with the positions of the lysine-arginine exchanges highlighted. (b) Analysis of iodixanol gradient-purified lysine AAV2 capsid exchange mutants by Western blotting using a SUMO1-specific antibody or the anti-VP specific antibody B1. (Top) K to R exchanges. (Bottom) K to Q exchanges for selected lysines.

Article Snippet: The membrane was blocked in 5% milk PBS-T at RT and then incubated with the desired primary antibody against SUMO1 (1:500; FL-101; Santa Cruz), SUMO2/3 (PA5-11373; 1:1,000; Sigma), DAXX (25c12, 1:1,000; CST), HA (1:500; sc-805; Santa Cruz), α-actin (1:5,000; MP), and VP (1:500 B1; Progen) in 5% PBS-T milk overnight at 4°C.

Techniques: Sequencing, Purification, Western Blot

Journal: Journal of Virology

Article Title: SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms

doi: 10.1128/JVI.00871-20

Figure Lengend Snippet: AAV2 K105R and K105Q capsid variants are SUMOylated. (a) Analysis of wt and mutant AAV capsid by Western blotting using a SUMO1- or VP-specific antibody. (b) Transduction of HeLa cells with wt and mutant AAV vectors. The y axis indicates relative light units. (c) Effect of SUMOylation knockdown on transduction efficiency of wt and mutant vectors.

Article Snippet: The membrane was blocked in 5% milk PBS-T at RT and then incubated with the desired primary antibody against SUMO1 (1:500; FL-101; Santa Cruz), SUMO2/3 (PA5-11373; 1:1,000; Sigma), DAXX (25c12, 1:1,000; CST), HA (1:500; sc-805; Santa Cruz), α-actin (1:5,000; MP), and VP (1:500 B1; Progen) in 5% PBS-T milk overnight at 4°C.

Techniques: Mutagenesis, Western Blot, Transduction, Knockdown

Journal: Journal of Biomedicine and Biotechnology

Article Title: Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization

doi: 10.1155/2011/320987

Figure Lengend Snippet: Antibody information.

Article Snippet: Sumo1 (FL-101) , Santa Cruz biotechnology , sc-9060 , 1 : 1000 , —.

Techniques: Ubiquitin Proteomics