MA1-26418

antibody from Invitrogen Antibodies

Targeting: RPA2

Provider product page for MA1-26418

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [41]
Comments [0]
Validations
Western blot [2]
Immunohistochemistry [1]
Other assay [10]

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Product number: MA1-26418 - Provider product page
Provider: Invitrogen Antibodies
Product name: RPA2 Monoclonal Antibody (9H8)
Antibody type: Monoclonal
Antigen: Other
Description: Recommended positive controls: Tonsil.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 9H8
Vial size: 500 µL
Storage: 4° C

RPA2 protein structure - MA1-26418 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 7. DDX11 facilitates resection and RPA loading to ssDNA substrates. ( A ) Quantification and representative micrographs of RPA32 foci in U2OS Ctrl and DDX11 KO cells recovering from an acute treatment with cisplatin (2.5 muM for 1 h) after 24 h. (Scale bar, 10 mum.) n = 2. Statistical analysis of foci was performed using Student''s t test. Error bars show average +- SD. ( B ) U2OS DIvA cells were transfected with siCtrl and si DDX11 followed by the addition of 4OHT (300 nM) to induce AsiSI-mediated DSBs after 48 h of post-transfection. Resection assay at the two different regions (KDELR3 and ASXL1) were analyzed after 4 h of 4OHT by real-time PCR. Values were normalized against the amount of ssDNA detected in control cells prior to 4OHT treatment, n >= 3. Corresponding Western blot and immunofluorescence for AsiSI-induced DSBs is shown. (Scale bar, 10 mum.) Statistical analysis was performed using Student''s t test. Error bars show average +- SEM. ( C ) Model for the DDX11 proposed role in resolving secondary structures upon DSB end resection to facilitate RPA loading and subsequently RAD51 nucleofilament formation required for homologous recombination-mediated DSB repair (see text for details).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. NSMF localizes to DNA lesions via the central residues of NSMF. ( A ) The kinetics of GFP-NSMF and mCherry-CDC5L recruitment to DNA lesions were examined by live cell imaging. The relative signal intensities are quantified and summarized in the bottom panel. The highest intensity on any lesion was set at 100% for each cell, and the recruitment kinetics were plotted. The average intensity for >=10 cells for each condition is presented graphically. Data represent the mean +- SD. ( B ) HeLa cells were treated with 1 muM CPT for 2 h or with 2 mM HU for 16 h and then harvested as whole-cell extracts (WCEs) or fractionated into soluble or chromatin-bound protein fractions. Each fraction was analyzed by western blot with the indicated antibodies. ( C ) HeLa cells transfected with GFP-NSMF expression plasmids were treated with 2 mM HU for 16 h and then incubated in fresh media for 3 h. Colocalization of GFP-NSMF and RPA2 was determined by immunofluorescence with an RPA2 antibody. Magnified insets show colocalization of NSMF and RPA2 foci. ( D ) HeLa cells were transfected with expression plasmids for GFP-NSMF WT or GFP-NMSF deletion mutants. After 24 h, the cells were laser microirradiated, and recruitment of GFP-NSMF proteins to DNA lesions was examined by live cell imaging. Relative intensities of the lesions were quantified, and the recruitment kinetics are plotted in the right panel. The average intensity for >=10 cells for each condition is presented graphically. Data

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. NSMF is involved in the replication stress response. ( A ) The NMSF gene was deleted from HeLa cells using CRISPR/Cas9. Expression of NSMF in WT and NSMF KO HeLa-cell extracts was analyzed by western blot. ( B - F ) Localization of RPA2 and phospho-RPA2 (S4/S8 or S32) was determined by immunofluorescence. NSMF WT and KO HeLa cells were treated with 2 mM HU for 16 h, fixed and stained with the indicated antibodies. RPA2 and phospho-RPA2 intensities per nucleus were quantified for each sample using ZEN Blue software (Carl Zeiss). The numbers above each sample indicate the number of nuclei analyzed. Data are presented as the mean +- SD; *** P < 0.001, **** P < 0.0001. ( G and H ) NMSF WT and KO HeLa cells were treated with either 2 mM HU for 16 h or 1 muM CPT for 2 h. The cells were fractionated, and phosphorylation of RPA2 in the chromatin-bound protein fraction was determined by western blot with the indicated antibodies. The levels of phospho-RPA2 (S4/S8 or S32) and total RPA were quantified using ImageJ software. Quantification of the western blots represents an average of three independent experiments. Data represent the mean +- SEM of three independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001. ( I and J ) Sensitivity of NSMF KO HeLa cells to DNA replication-blocking agents. NSMF WT and KO HeLa cells were plated and treated with the indicated concentrations of HU or CPT. The numbers of surviving colonies were counted 14 days after treatment. Data repr

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. NSMF directs recruitment of ATRIP-ATR to DNA lesions by mediating the interaction between RPA2 and ATRIP-ATR. ( A ) NSMF WT and KO HeLa cells were transfected with GFP-ATRIP expression plasmids. After 24 h, the cells were microirradiated, and recruitment of GFP-ATRIP to DNA lesions was examined by live cell imaging. ( B ) NSMF WT and KO HeLa cells were treated with or without 2 mM HU for 16 h. The cells were fractionated, and the chromatin-bound protein fraction was subjected to western blot with the indicated antibodies. ( C ) Cotransfection of GFP-ATRIP with the mCherry-vec, mCherry-NSMF-WT or mCherry-NSMF-D4 mutant plasmid in NSMF KO#1 and KO#2 HeLa cells. After 24 h, the transfected cells were microirradiated, and recruitment of ATRIP and NSMF to DNA lesions was examined by live cell imaging. ( D and E ) HeLa-cell lysates were immunoprecipitated with anti-IgG, anti-ATRIP or anti-RPA2 antibodies. Endogenous interactions between NSMF and ATRIP or RPA2 were determined by western blot with the indicated antibodies. ( F and G ) HEK293T cells were cotransfected with a Flag-NSMF-deletion mutant and either GFP-ATRIP or Myc-RPA2. Immunoprecipitation using an anti-FLAG antibody and western blot with the indicated antibodies were performed to identify the NSMF regions that interact with ATRIP (F) or RPA2 (G). ( H ) HEK293T cells were transfected with GFP-ATRIP or Myc-RPA2 and either FLAG-NSMF WT or FLAG-NSMF D2. Immunoprecipitation with an anti-FLAG antibody and western bl

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6. NSMF mediates RPA2 ubiquitination. ( A ) Endogenous ATR in NSMF WT or KO HeLa cell lysates was immunoprecipitated with anti-ATR antibody and western blotting with the indicated antibodies was performed. ( B ) NSMF WT and KO HeLa cells were transfected with Myc-RAP2 and HA-ubiquitin and were treated with or without 2 mM HU for 3 h. Cells were lysed in denaturing conditions, diluted and ubiquitinated proteins were immunoprecipitated with anti-HA antibody and subjected to western blotting with the indicated antibodies. ( C ) NSMF WT and KO HeLa cells transfected with HA-ubiquitin were treated with or without 2 mM HU for 3 h. Cells were lysed in denaturing conditions, diluted, and ubiquitinated proteins were immunoprecipitated with anti-HA antibody. Endogenous RPA2 ubiquitination was detected by immunoblotting with the indicated antibodies. ( D ) HeLa cells transfected with HA-ubiquitin and Flag-NSMF were treated with or without 2 mM HU for 3 h. Cells were lysed in denaturing conditions, diluted, ubiquitinated proteins were immunoprecipitated with anti-HA antibody, and RPA2 ubiquitination was detected by immunoblotting with the indicated antibodies. ( E ) HeLa cells transfected with Myc-RPA2 and Flag-NSMF, plus the WT, lysine mutant K48R, or K63R form of HA-ubiquitin, were treated with or without 2 mM HU for 3 h. Cells were lysed in denaturing conditions, diluted and ubiquitinated proteins and immunoprecipitated with anti-HA antibody. RPA2 ubiquitination was detected by

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7. NSMF participates in genome maintenance and mouse survival in response to in vivo DNA damage. ( A ) Diagram describing generation of the Nsmf KO allele using a gene targeting vector. The vector consists of a Flp-recombinase target (FRT)-flanked selection cassette inserted between the third and fourth exons of Nsmf . The Nsmf transcript is trapped through the splice acceptor (SA) element and truncated through the SV40 polyadenylation signal (pA). lacZ, beta-galactosidase; Neo, Neomycin resistance gene. The red arrow is the primer for genotyping. ( B ) Genotyping of Nsmf +/+, Nsmf +/- and Nsmf -/- mice using tail DNA. The sizes of the PCR products for the WT and mutant alleles are 343 and 292 bp, respectively. ( C and D ) Confirmation of Nsmf removal by qRT-PCR (C) and western blot analysis (D) using MEF mRNA and protein lysates, respectively. ( E ) Impaired interaction between RPA2 and ATRIP in NSMF KO MEF cells. Endogenous RPA2 in NSMF WT and KO MEF cell lysates was immunoprecipitated with anti-RPA2 antibody, and western blotting with the indicated antibodies was performed. ( F and G ) NSMF WT or KO cells were treated with 2 mM HU for 24 h, transferred to fresh media and subjected to metaphase spreading. (F) Representative images of spontaneous and HU-induced chromosome instability in NSMF WT and KO MEFs. The red dots indicate chromosomal abnormalities, such as chromosome breakage, fragments and radial figures. (G) The numbers of abnormal chromosomes per cell were p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. RPA32 ubiquitylation is regulated by PI3K-like kinases. (A) Cells were transfected with a vector expressing Strep-HA-tagged ubiquitin, pre-treated for 1 h with VE-821 (ATRi, 10 muM), KU55933 (ATMi, 10 uM), NU7441 (DNAPKi, 2 muM) or a combination of all three inhibitors, treated with 1 muM CPT for 4 h and lysed under denaturing conditions. Strep-Tactin pulldown was performed to isolate ubiquitylated proteins. ( B ) Cell transfection with an SFB-PRP19 vector were treated as in A and streptavidin pulldown was performed to isolate PRP19 along with its interactors. Controls for the efficiency of the inhibitor treatments are provided in ( S2A ). ( C and D ) U2OS cells were pre-sensitized with 10 muM BrdU, treated with the indicated inhibitors along with DRB for 1 h and damaged by UV-laser microirradiation. Recruitment of PRP19 and RPA32 was monitored 2 h after damage by immunofluorescence.