sirf mouse anti-rad51c 2h11/6 novus biologicals nb100-177  (Novus Biologicals)

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Evaluation of 173 RAD51C missense variants by HDR assay. A, DR-GFP reporter assay showing the range of HDR activity for 173 missense variants in CL-V4B cells, measured as fold change in GFP-positive cells (normalized to 1–5 scale, WT = 5 and p.Leu138Phe = 1). Neutral (>2.5 scale, gray bars), deleterious (<1.25 scale, red bars), and intermediate effects (>1.25, <2.5 scale, light gray bars). Purple, L138F and C135Y deleterious controls. Amino acid changes in one letter code are labeled on the x -axis in clusters (black, blue, and orange) in order of presentation on the bar chart. Error bars, SEM of three independent experiments. B, Illustration of the location of missense variants within the RAD51C linear sequence identifying a deleterious variant hotspot. Key functional domains of RAD51C are indicated and neutral (blue), deleterious (orange), and intermediate (green) variants are shown at top. C, Circos plot of the RAD51C variants and functional assay (HDR, cisplatin and olaparib sensitivity, binding to XRCC3, RAD51D, and XRCC2) results. RAD51C variants are indicated by residue position in the outer ring. Track 1 shows the final score based on all functional assays. For HDR, variants were classified as neutral (light blue; ≥51.1% relative to WT), intermediate (green; 48.7%–26.8%), or deleterious (orange; ≤22.7%). Cisplatin sensitivity was classified as neutral (≥83.5% relative to WT), intermediate (43%–82%), or deleterious (≤13.1%). Olaparib sensitivity was classified as neutral (≥ 80.4% relative to WT), intermediate (78.7%–51.7%), or deleterious (≤42%). Dark blue, interactions with XRCC3, RAD51D, and XRCC2; yellow, partial interaction; red, no interaction.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: Reporter Assay, Activity Assay, Labeling, Sequencing, Variant Assay, Functional Assay, Binding Assay, Residue

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Influence of RAD51C missense variants on response to cisplatin and olaparib treatment. Relative IC 50 values (normalized to 1–15 scale, WT = 15 and p.Cys135Tyr = 1) from an MTS assay of CL-V4B cells, transduced with selected RAD51C variant lentivirus, 5 days after treatment with varying doses of cisplatin ( A ) and olaparib ( B ). Variants defined as neutral (gray), intermediate (light gray), and deleterious (red) by the HDR assay are shown. Purple, L138F and C135Y deleterious controls. Error bars, SEM of three independent experiments.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: MTS Assay, Transduction, Variant Assay

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Drug sensitivity of human U2OS landing pad cells. A, Olaparib sensitivity associated with RAD51C variants. Cell survival of U2OS cells expressing RAD51C variants was quantified after 4 days of treatment and calculated relative to mock-treated cells. Mean ± SEM was calculated from three independent experiments, each performed in triplicate. B, RAD51C protein levels in stable U2OS landing pad cell lines. Expression of RAD51C WT and variant proteins in U2OS cells was determined by Western blot analysis after 24 hours of doxycycline induction.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: Expressing, Variant Assay, Western Blot

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Characterization of U20S landing pad cells expressing RAD51C variants. A, Colony formation assays of RAD51C variants. B, RAD51 foci quantification and representative microscopy images for each variant. RAD51 foci formation was quantified after exposure of landing pad cells to 5 Gy of γ-irradiation. Each dot represents a geminin (S/G 2 phase)-positive cell and the bars designate the mean number of foci in at least 1,000 cells obtained in at least three independent experiments. The mean change percentage was calculated for each variant relative to the WT.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: Expressing, Microscopy, Variant Assay, Irradiation

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Coimmunoprecipitation analysis of RAD51C CX3 and BCDX2 complexes. Western blotting of coimmunoprecipitated Flag-tagged RAD51C variant proteins with XRCC3 and RAD51D, XRCC2 paralogs 48 hours after transfection of HEK293T cells with FLAG-tagged RAD51C variant expression plasmids. IP, immunoprecipitation; TCL, total cell lysate.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: Western Blot, Variant Assay, Transfection, Expressing, Immunoprecipitation

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Evaluation of RAD51C deleterious variants in RAD51C 3D structure prediction model. A, Locations of 30 neutral and 30 deleterious variants in the 3D model of two ATP-bound RAD51C monomers (yellow and green). The locations of neutral and deleterious variants are shown with green and red dots, respectively. B, Magnified view of the 11 deleterious variants predicted to disrupt the binding of ATP.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: Binding Assay

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Association of functionally characterized RAD51C variants with breast and ovarian cancer risk.

Article Snippet: RAD51C expression was assessed by Western blotting with anti-RAD51C mouse mAb (mAb F11; Santa Cruz Biotechnology) and anti-FLAG mouse monoclonal (mAB M2; Sigma; ref. ).

Techniques: Variant Assay, Control