Journal: Nucleic Acids Research

Article Title: Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

doi: 10.1093/nar/gkn895

Figure Lengend Snippet: Photo-cross-linking reactions between hRPA and Oligo 31S and identification of complexes by immunoblotting. ( A ) 32 P-labeled Oligo 31S (2 nM) were mixed with the indicated amounts of hRPA then irradiated at 15°C for 45 min. SDS-PAGE (10%) were used to separate multiple cross-linked species. Cross-linked species (arrows) were detected and quantified by the phosphorimager—Molecular Dynamic. ( B ) After electrophoresis and transfer onto a nitrocellulose membrane, DNA was removed by incubation of the membrane with DNase I. ECL western blotting used anti-RPA1 (AbRPA1), anti-RPA2 (AbRPA2) and anti-RPA3 (AbRPA3) antibodies to reveal the cross-linked RPA1, RPA2 and RPA3 subunits, respectively. The immunoblot shown here identifies each subunit implicated in photo-cross-linking with Oligo 31S 29 and 10 nM of hRPA (lanes 1). For comparison, free hRPA (10 nM) was loaded (lanes 2).

Article Snippet: Rabbit anti-RPA1 polyclonal antibodies were obtained from Chemicom International.

Techniques: Western Blot, Labeling, Irradiation, SDS Page, Electrophoresis, Membrane, Incubation, Comparison

Journal: Nucleic Acids Research

Article Title: Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

doi: 10.1093/nar/gkn895

Figure Lengend Snippet: Quantification of cross-linked species. The relative amounts of cross-linked species involving RPA1 (red, long dashed line), RPA2 (blue, medium dashed line) and RPA3 (green, short dashed line) subunits were quantified for each hRPA concentration from the hRPA-Oligo 31S gel ( A). Error bar is ±20%. The results obtained for 2 nM of hRPA were not reported since no cross-links were observed except for Oligo 31S 29 . To correlate the nature of the complexes with the presence of the subunit on the DNA, the relative yield of each complex was taken from data in B. The same scales are used for each graph, but to simplify the figure only scales for the Oligo 31S 3 graph are shown.

Article Snippet: Rabbit anti-RPA1 polyclonal antibodies were obtained from Chemicom International.

Techniques: Concentration Assay

Journal: Nucleic Acids Research

Article Title: Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

doi: 10.1093/nar/gkn895

Figure Lengend Snippet: Schematic models of the 30, 13–22 and 8–10 nt binding modes. The s 4 T substituted positions are represented by dashed lines. RPA1 is represented by a red bubble, RPA2 by a blue bubble and RPA3 by a green bubble.

Article Snippet: Rabbit anti-RPA1 polyclonal antibodies were obtained from Chemicom International.

Techniques: Binding Assay

Journal: BMC Cancer

Article Title: Potential of extravasated platelet aggregation as a surrogate marker for overall survival in patients with advanced gastric cancer treated with preoperative docetaxel, cisplatin and S-1: a retrospective observational study

doi: 10.1186/s12885-017-3279-4

Figure Lengend Snippet: Univariate/multivariate analyses of factors that are reportedly associated with chemoresistance in the p-DCS group

Article Snippet: Subsequently, sections were incubated for 2 h at room temperature with a 1:100 diluted anti-platelet antibody (anti-CD42b rabbit monoclonal, EPR6995; Abcam, Tokyo, Japan); a 1:50 diluted anti-podoplanin antibody (anti-D2–40 mouse monoclonal, Code IR072/IS072; Dako, Tokyo, Japan); a 1:50 diluted anti-forkhead box (FOX)P3 antibody (anti-FOXP3 mouse monoclonal, 236A/E7; Abcam), and a 1:50 diluted anti-SNAIL antibody (anti-SNAIL rabbit polyclonal antibody, ab180714; Abcam).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Potential of extravasated platelet aggregation as a surrogate marker for overall survival in patients with advanced gastric cancer treated with preoperative docetaxel, cisplatin and S-1: a retrospective observational study

doi: 10.1186/s12885-017-3279-4

Figure Lengend Snippet: Univariate/multivariate analyses of factors associated with prognosis in the p-DCS group

Article Snippet: Subsequently, sections were incubated for 2 h at room temperature with a 1:100 diluted anti-platelet antibody (anti-CD42b rabbit monoclonal, EPR6995; Abcam, Tokyo, Japan); a 1:50 diluted anti-podoplanin antibody (anti-D2–40 mouse monoclonal, Code IR072/IS072; Dako, Tokyo, Japan); a 1:50 diluted anti-forkhead box (FOX)P3 antibody (anti-FOXP3 mouse monoclonal, 236A/E7; Abcam), and a 1:50 diluted anti-SNAIL antibody (anti-SNAIL rabbit polyclonal antibody, ab180714; Abcam).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Defective membrane repair machinery impairs survival of invasive cancer cells

doi: 10.1038/s41598-020-77902-5

Figure Lengend Snippet: Expression and subcellular localization of endogenous Anx in MDA-MB-231. ( a ) 10 µg of protein extracts from MDA-MB-231 and BeWo cells were analyzed by western-blot. The gel analysis plugin of ImageJ was used to quantify the density of bands. The histogram presents mean values (± SD) of the ratio Anx/GAPDH from ten independent experiments. Student t Test for independent samples. ** p < 0.01. ( b ) Revelation of AnxA5 in MDA-MB-231 and BeWo cells compared to 50 ng purified recombinant AnxA5 (Pure A5), presented as an example. The whole membrane, together with representative western-blot analysis for AnxA1, A2, A4 and A6, are presented in Supplementary Fig. . ( c ) MDA-MB-231 cells were immunostained for AnxA1, AnxA2, AnxA4, AnxA5 and AnxA6 (green), as indicated, and counterstained with DAPI (blue). The right-hand column presents magnified images extracted from the “Anx” column (indicated by inserts). Scale bar: 40 µm. ( d ) Quantification of fluorescence intensities after immunostaining of endogenous Anx in MDA-MB-231 cells. Fluorescence intensities were measured from 8-bit images by drawing a circular ROI inside the cytoplasm and measuring the mean pixel value. Mean pixel values from at least 20 cells are presented. Horizontal bars represent median values. The variance value is 323, 268, 224, 2372 and 2909 for AnxA1, A2, A4, A5 and A6, respectively. The coefficient of variation is 14%, 13%, 24%, 35% and 47% for AnxA1, A2, A4, A5 and A6, respectively. The expression level of AnxA5 or AnxA6 is heterogenous from one MDA-MB-231 cell to another. In some cells, AnxA5 and AnxA6 are expressed at a very low level.

Article Snippet: Semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content in AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA4 (32 kDa), AnxA5 (35 kDa), AnxA6 (68 kDa), and glyceraldehyde-3-phosphate deshydrogenase (GAPDH, loading control, 37 kDa) was detected with rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA4 monoclonal antibody (SAB4200121, Sigma), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-AnxA6 monoclonal antibody (sc-271859, Santa cruz Biotechnology), and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Expressing, Western Blot, Purification, Recombinant, Membrane, Fluorescence, Immunostaining