Journal: Journal of Virology

Article Title: ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment

doi: 10.1128/JVI.01748-17

Figure Lengend Snippet: Antibodies used for NK proliferation panel

Article Snippet: KIR3DLO1 , NKVFS1 , PE , 130-092-688 , Miltenyi.

Techniques:

Journal: Journal of Virology

Article Title: ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment

doi: 10.1128/JVI.01748-17

Figure Lengend Snippet: Antibodies used for NK functional assay

Article Snippet: KIR3DLO1 , NKVFS1 , PE , 130-092-688 , Miltenyi.

Techniques: Functional Assay

Journal: Structure (London, England : 1993)

Article Title: Magnesium Activates Microsecond Dynamics to Regulate Integrin-Collagen Recognition

doi: 10.1016/j.str.2018.05.010

Figure Lengend Snippet: Key Resources Table

Article Snippet: All software are listed in the and the use of each package for data analysis is described in the sub-headings of STAR Methods. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Integrin α1 antibody, clone FB12 Millipore EMD Cat#MAB1973; RRID: AB_2129087 Goat anti-mouse IgG HRP antibody Genescript Corporation Cat#NC1348387; RRID: AB_1968937 Bacterial and Virus Strains E. coli BL21(DE3) competent cells Novagen Cat#69450 Chemicals, Peptides, and Recombinant Proteins Synthetic collagen model peptide (Ac-(GPO) 4 GLOGEN(GPO) 4 GY-NH 2 ) LifeTein N/A Recombinant protein: human α 1 I (aa 141-335, ref# {"type":"entrez-protein","attrs":{"text":"NP_852478.1","term_id":"31657142"}} NP_852478.1 ) This paper N/A Recombinant protein: human α 1 I (aa 141-331, ref# {"type":"entrez-protein","attrs":{"text":"NP_852478.1","term_id":"31657142"}} NP_852478.1 ) ( Nunes et al., 2016 ) N/A AccuPrime™ Pfx DNA Polymerase Fisher Scientific Cat#12-344-024 DpnI New England Biolabs Cat#R0176S Collagen I from rat tail BD Biosciences Cat#354236 Albumin Bovine (BSA) Fraction V 10 VWR Cat#97061-416 Critical Commercial Assays Pierce™ TMB Substrate Kit ThemoFisher Scientific Cat#34021 Deposited Data Relaxation dispersion data Datasets in , , , S4 and S6 .

Techniques: Recombinant, Plasmid Preparation, Software

Journal:

Article Title: BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

doi: 10.1242/jcs.051813

Figure Lengend Snippet: BLM and RAD54 have in vivo functional interaction. (A) Loss of BLM enhances the number of RAD54 foci. BS/A-15 cells were either left untreated or treated with HU for 12 hours. Extent of RAD54 foci formation was determined by immunofluorescence (top) and quantified (bottom). (B) BLM, RAD51 and RAD54 colocalize in vivo. IF with RAD51, BLM and RAD54 antibodies was carried out in HU-treated NHFs. Scale bars: 5 μm. (C) BLM and RAD54 interact in vivo. Immunoprecipitation was carried out with nuclear extracts from NHFs with antibodies against either BLM or RAD54 and probed with indicated antibodies.

Article Snippet: His-tagged RAD51 was purchased from Calbiochem. pGEX4T-1 RAD54 constructs were generated by cloning the respective PCR products into: (1) Not I site for pGEX4T-1 RAD54(1-747), (2) Bam H1- Xho I sites for pGEX4T-1 RAD54(243-747), pGEX4T-1 RAD54(485-625), pGEX4T-1 RAD54(626-748), pGEX4T-1 RAD54(668-748); (3) Bam H1- Eco RI sites for pGEX4T-1 RAD54(1-155) and pGEX4T-1 RAD54(156-344). pRS-shRNA RAD54 constructs (TR309964) were purchased from Origene.

Techniques: In Vivo, Functional Assay, Immunofluorescence, Immunoprecipitation

Journal:

Article Title: BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

doi: 10.1242/jcs.051813

Figure Lengend Snippet: Internal residues of BLM interact with N- and C-termini of RAD54. (A) Summary of BLM-RAD54 interactions (left). Coomassie-blue-stained gel showing the expression of purified GST or GST-tagged BLM fragments BLM(1-212), BLM(191-660), BLM(621-1041), BLM(1001-1417), BLM(1-1417) and BLM(1-1417) K695A (right). (B) N-terminal region of bound GST-tagged BLM interacts with 35S-labeled full-length RAD54. After pull-down, bound radioactive RAD54 was determined by autoradiography. (C) A 10 amino acid stretch in the N-terminus of BLM interacts with RAD54. Bound GFP-RAD54 was incubated with soluble BLM fragments. After pull-down, the blots were probed with anti-GST and anti-GFP antibody. Asterisk indicates a crossreactive band migrating at the same molecular mass as BLM 1-212. (D) Coomassie-blue-stained gel showing the expression of purified GST or GST-tagged RAD54 fragments. Bottom gel shows that N- and C-terminal domains of RAD54 are required for its interaction with BLM. 35S-labeled full-length BLM was incubated with wild-type RAD54 or its various fragments.

Article Snippet: His-tagged RAD51 was purchased from Calbiochem. pGEX4T-1 RAD54 constructs were generated by cloning the respective PCR products into: (1) Not I site for pGEX4T-1 RAD54(1-747), (2) Bam H1- Xho I sites for pGEX4T-1 RAD54(243-747), pGEX4T-1 RAD54(485-625), pGEX4T-1 RAD54(626-748), pGEX4T-1 RAD54(668-748); (3) Bam H1- Eco RI sites for pGEX4T-1 RAD54(1-155) and pGEX4T-1 RAD54(156-344). pRS-shRNA RAD54 constructs (TR309964) were purchased from Origene.

Techniques: Staining, Expressing, Purification, Labeling, Autoradiography, Incubation

Journal:

Article Title: BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

doi: 10.1242/jcs.051813

Figure Lengend Snippet: BLM prevents RAD54-RAD51 complex formation in vitro and in vivo. (A) Purified proteins used to test the BLM(1-212)-mediated prevention of RAD51-RAD54 complex formation. (B,C) BLM prevents RAD54-RAD51 interaction. Interaction between bound GFP-RAD54 and soluble His-RAD51 was determined in the absence or presence of soluble BLM(1-212) or GST. The levels of the respective proteins after interaction were assessed by western blotting (B). Soluble BLM(109-212) was also used (C). (D) BLM prevents RAD51-RAD54 complex formation in vivo. Immunoprecipitation was carried out on the nuclear extract from BS/A-15 cells with antibodies against RAD51 or RAD54. Western blots were then performed with same antibodies. (E) BLM decreases colocalization between RAD51 and RAD54. Immunofluorescence of BS/A-15 cells with antibodies against RAD51 and RAD54. Scale bars: 5 μm.

Article Snippet: His-tagged RAD51 was purchased from Calbiochem. pGEX4T-1 RAD54 constructs were generated by cloning the respective PCR products into: (1) Not I site for pGEX4T-1 RAD54(1-747), (2) Bam H1- Xho I sites for pGEX4T-1 RAD54(243-747), pGEX4T-1 RAD54(485-625), pGEX4T-1 RAD54(626-748), pGEX4T-1 RAD54(668-748); (3) Bam H1- Eco RI sites for pGEX4T-1 RAD54(1-155) and pGEX4T-1 RAD54(156-344). pRS-shRNA RAD54 constructs (TR309964) were purchased from Origene.

Techniques: In Vitro, In Vivo, Purification, Western Blot, Immunoprecipitation, Immunofluorescence

Journal:

Article Title: BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

doi: 10.1242/jcs.051813

Figure Lengend Snippet: Lack of BLM leads to a decrease in the dynamic mobility of RAD51. (A) RAD51 is present in the soluble fraction in the presence of BLM. Nuclei of BS/A-15 cells were subfractionated into RNP-enriched (fraction III) and soluble (fraction I). Levels of BLM, RAD54 and RAD51 were determined by western blotting. (B) FRAP recovery curves of GFP-RAD51 in BS/A-15 cells. The recovery curves were obtained for GFP-RAD51 present in nucleoplasm or foci. Each data point represented the mean of a number of cells (indicated on right). t1/2 represents half maximal recovery time. (C) FRAP recovery curves of BS/A-15 cells transfected with GFP-RAD54. (D) FRAP recovery curves on of SV40-immortalized GM08505 cells stably expressing GFP-BLM (GFP-BLM).

Article Snippet: His-tagged RAD51 was purchased from Calbiochem. pGEX4T-1 RAD54 constructs were generated by cloning the respective PCR products into: (1) Not I site for pGEX4T-1 RAD54(1-747), (2) Bam H1- Xho I sites for pGEX4T-1 RAD54(243-747), pGEX4T-1 RAD54(485-625), pGEX4T-1 RAD54(626-748), pGEX4T-1 RAD54(668-748); (3) Bam H1- Eco RI sites for pGEX4T-1 RAD54(1-155) and pGEX4T-1 RAD54(156-344). pRS-shRNA RAD54 constructs (TR309964) were purchased from Origene.

Techniques: Western Blot, Transfection, Stable Transfection, Expressing

Journal:

Article Title: BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

doi: 10.1242/jcs.051813

Figure Lengend Snippet: BLM stimulates RAD54 function. (A) The N-terminal 1-212 amino acids of BLM enhance RAD54-mediated ATPase activity. ATPase activity was assessed with different combinations of proteins, as indicated. The concentrations of each protein are indicated in the Materials and Methods. *P<0.05 compared with the ATPase activity obtained by RAD54 alone. (B) Loss of ATPase activity in BLM(1-1417) K695A mutant. ssDNA-dependent ATPase activity was assessed using BLM(1-1417) or BLM(1-1417) K695A. (C) ATPase-dead mutant of BLM stimulates RAD54-mediated ATPase activity. BLM(1-1417) K695A was used to determine its effect on RAD54-mediated ATPase activity. (D) Schematic diagram of G5E4 array (Zhang et al., 2007). Representative REA assays with RAD54 alone (±ATP), RAD54+BLM(1-212) (+ATP) and BLM(1-212) alone (+ATP). (E) Quantification of results in D and of REA assays involving RAD54 and BLM(1-1417) K695A (+ATP).

Article Snippet: His-tagged RAD51 was purchased from Calbiochem. pGEX4T-1 RAD54 constructs were generated by cloning the respective PCR products into: (1) Not I site for pGEX4T-1 RAD54(1-747), (2) Bam H1- Xho I sites for pGEX4T-1 RAD54(243-747), pGEX4T-1 RAD54(485-625), pGEX4T-1 RAD54(626-748), pGEX4T-1 RAD54(668-748); (3) Bam H1- Eco RI sites for pGEX4T-1 RAD54(1-155) and pGEX4T-1 RAD54(156-344). pRS-shRNA RAD54 constructs (TR309964) were purchased from Origene.

Techniques: Activity Assay, Mutagenesis

Journal:

Article Title: BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

doi: 10.1242/jcs.051813

Figure Lengend Snippet: Model depicting the regulation of homologous recombination by BLM as a result of alteration of RAD51 and RAD54 function. Double-strand breaks (DSBs) are recognized by the HR machinery after the chromatin-remodeling phase. BLM enhances the ATPase activity of the chromatin remodeler RAD54, thereby enhancing its remodeling activity in a homology-independent manner. Subsequently, BLM dissociates from RAD54, allowing the same region to instead bind to RAD51. The next stage of RAD54-driven chromatin remodeling is possibly homology driven and stimulated by RAD51 bound to single-stranded DNA (RAD51-ssDNA). The result of chromatin remodeling allows the sequential accumulation of proteins during subsequent stages of HR. After detection of DSB and resection of the DNA in the 5′-3′ direction, RAD51 binds to ssDNA and displaces replication protein A (RPA), which leads to RAD51 polymerization (this phase is referred to as the presynaptic phase). RAD54 promotes the nucleation of RAD51 on the RPA-coated ssDNA, thereby initiating the presynaptic phase of HR. Once the homology search is successful, the duplex is captured and the RAD51 filament invades it to form the heteroduplex structure (synaptic phase). RAD54 stabilizes the RAD51-ssDNA complex, thereby promoting this process. At synaptic phase BLM(possibly phosphorylated by ATR at Thr99) interacts with RAD51 and disrupts RAD51 filaments. Heteroduplex DNA extension and branch migration normally occurs during the postsynaptic phase of HR. DNA polymerases use the intact copy to re-synthesize the deleted DNA sequences, DNA ligases join the newly synthesized fragments and the Holliday junctions are resolved by specific endonucleases, known as resolvases. As a result of the above two activities, BLM can accurately control HR. Additional mechanistic processes, which BLM is known to use at late stages of HR, are not shown.

Article Snippet: His-tagged RAD51 was purchased from Calbiochem. pGEX4T-1 RAD54 constructs were generated by cloning the respective PCR products into: (1) Not I site for pGEX4T-1 RAD54(1-747), (2) Bam H1- Xho I sites for pGEX4T-1 RAD54(243-747), pGEX4T-1 RAD54(485-625), pGEX4T-1 RAD54(626-748), pGEX4T-1 RAD54(668-748); (3) Bam H1- Eco RI sites for pGEX4T-1 RAD54(1-155) and pGEX4T-1 RAD54(156-344). pRS-shRNA RAD54 constructs (TR309964) were purchased from Origene.

Techniques: Homologous Recombination, Activity Assay, Migration, Synthesized

Journal: Journal of Translational Medicine

Article Title: HLA-A24 and survivin: possibilities in therapeutic vaccination against cancer

doi: 10.1186/1479-5876-4-38

Figure Lengend Snippet: Detection of HLA-A24 restricted, sur20–28 specific, CD8 postitive cells by interferon-γ and perforin . ELISPOT. CD8+ cells were isolated from PBL from two HLA-A24+ breast cancer patients (c24, c29), two HLA-A24+ melanoma patients (m3, m6), and four HLA-A24+ renal cancer patients (u1, u8, u13, u15). Spontaneous T-cell responses against Sur20–28 was measured by both interferon-γ and perforin ELISPOT for all patients. The average number of peptide specific IFNγ or perforin spots formed in response to Sur20–28 among 10 5 in vitro stimulated CD8+ cells. Non-specific spots are subtracted.

Article Snippet: After incubation (37°C/5%CO 2 ) medium was discarded and the wells were washed prior to addition of the secondary detection Ab, anti-human biotinylated interferon-γ (Mabtech) at 2 μg/ml in 75 μl PSB/BSA per well or anti-human biotinylated perforin (Pf-344-biotin, Mabtech) at 1 μg/ml in 100 μl PSB, respectively.

Techniques: Enzyme-linked Immunospot, Isolation, In Vitro