rabbit anti rnf8  (Novus Biologicals)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear-Localized BCKDK Facilitates Homologous Recombination Repair to Support Breast Cancer Progression and Therapy Resistance.

doi: 10.1002/advs.202416590

Figure Lengend Snippet: Figure 2. Branched-chain 𝛼-ketoacid dehydrogenase kinase (BCKDK) binds with and phosphorylates RNF8 at S157 to modulate homologous recombi- nation repair (HRR). a) Immunoprecipitation-mass spectrometry (IP-MS) analysis of nuclear proteins interacting with BCKDK in Flag-BCKDK overex- pressed MDA-468 cells. The top five rated DNA damage repair proteins are listed. b) Co-IP assay in MDA-468 cells co-transfected with HA-BCKDK and Flag-tagged RNF8. Cell lysates were immunoprecipitated with Flag antibody, followed by Western blot analysis with antibodies against HA or Flag. c) In vitro kinase assay analysis of GST-RNF8 phosphorylation in the absence or presence of His-BCKDK by adding ATP at the indicated concentrations. d) MDA-468 cells stably expressing nontargeting control vectors (NTC) or BCKDK shRNAs were transfected with Flag-RNF8WT, Flag-RNF8S157A, or Flag-RNF8S157D vectors. Cells were harvested and subjected to IP with Flag antibody, followed by Western blot to detect p-RNF8S157, Flag, and BCKDK levels. e) Immunofluorescence (IF) staining with 𝛾H2A.X foci (red) and DAPI (blue) in MDA-468 cells stably expressing NTC or RNF8 shRNAs, followed by transfected with RNF8WT, RNF8S157A, RNF8S157D, or control vectors. Representative images are shown on the left, with the number of foci per cell quantified on the right top, Western blot analysis of RNF8 and p-RNF8S157 was shown on the right bottom. Scale bar, 5 μm. f) HRR efficiency levels were tested via I-Scel HRR system in U2OS cells stably expressing NTC or RNF8 shRNAs, followed by transfected with RNF8WT, RNF8S157A, RNF8S157D, or control vectors. g) IF staining with 𝛾H2A.X foci (red) and DAPI (blue) in MDA-468 cells stably expressing BCKDK and NTC or RNF8 shRNAs, followed

Article Snippet: Primary antibodies were used against the following proteins: BCKDK (Santa Cruz, sc-374424), RNF8 (Abcam, ab234977), p-RNF8S157 (Abclonal, E19977), RAD51 (Proteintech, 67024-1-Ig), Ku70 (Proteintech, 10723-1-AP), p-RPA32 (Bethyl, A300-245A-M), RPA32 (Proteintech, 10412-1-AP), γH2A.X (BBI, D551270025; CST, 9718S), H2A.X (Proteintech, 10856-1-AP), RAD50 (Sangon Biotech, D160725-0025), RAD52 (Sangon Biotech, D160724-0025), pBCKDHA (Abcam, ab200577), Flag-tag (Sigma, F1804), and β-actin (Proteintech, 66009-1-Ig).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Western Blot, In Vitro, Kinase Assay, Phospho-proteomics, Stable Transfection, Expressing, Control, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Nuclear-Localized BCKDK Facilitates Homologous Recombination Repair to Support Breast Cancer Progression and Therapy Resistance.

doi: 10.1002/advs.202416590

Figure Lengend Snippet: Figure 5. Aberrant branched-chain 𝛼-ketoacid dehydrogenase kinase (BCKDK)/p-RNF8S157/RNF8 axis predicts breast cancer progression and mortality. a) Western blot analysis of BCKDK, p-RNF8S157, RNF8, and RAD51 levels in 14 pairs of clinically matched with tumor-adjacent noncancerous tissues (N) and breast cancer tissues (T). b) Representative immunohistochemistry (IHC) images showing BCKDK, p-RNF8S157, or RNF8 levels in normal breast tissue (normal) and breast cancer specimens of different clinical stages (I-III). Scale bars, 50 μm. Insets, fourfold magnification; scale bars, 20 μm. c) Statistical quantification of the mean intensity of BCKDK (c, left), and p-RNF8S157(c, right) staining using HistoQuest software (healthy donors (N),

Article Snippet: Primary antibodies were used against the following proteins: BCKDK (Santa Cruz, sc-374424), RNF8 (Abcam, ab234977), p-RNF8S157 (Abclonal, E19977), RAD51 (Proteintech, 67024-1-Ig), Ku70 (Proteintech, 10723-1-AP), p-RPA32 (Bethyl, A300-245A-M), RPA32 (Proteintech, 10412-1-AP), γH2A.X (BBI, D551270025; CST, 9718S), H2A.X (Proteintech, 10856-1-AP), RAD50 (Sangon Biotech, D160725-0025), RAD52 (Sangon Biotech, D160724-0025), pBCKDHA (Abcam, ab200577), Flag-tag (Sigma, F1804), and β-actin (Proteintech, 66009-1-Ig).

Techniques: Western Blot, Immunohistochemistry, Staining, Software

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: Key Resources Table

Article Snippet: Rabbit polyclonal anti-RNF8 , Proteintech , Cat#14112-1-AP.

Techniques: Recombinant, In Situ, Sequencing, Software