Journal: Antioxidants

Article Title: Treatment of Human HeLa Cells with Black Raspberry Extracts Enhances the Removal of DNA Lesions by the Nucleotide Excision Repair Mechanism

doi: 10.3390/antiox11112110

Figure Lengend Snippet: Enhancement of NER protein expression in BRBE-treated HeLa cells. ( A ) XPA, XPB and ( B ) XPD protein expression in HeLa cells, all with and without pre-treatment with BRBE. Tubulin is an inert cellular protein that was used as a control for the overall cellular protein concentrations in BRBE-treated and untreated cells. All Western Blot measurements of relative protein concentrations were conducted within the linear range of overall protein concentrations shown in .

Article Snippet: The blots were stained with XPA (sc-28353), XPB (sc-271500), anti -XPD (sc-101174), and anti -Tubulin (sc-53140) primary monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Control, Western Blot

Journal: Antioxidants

Article Title: Treatment of Human HeLa Cells with Black Raspberry Extracts Enhances the Removal of DNA Lesions by the Nucleotide Excision Repair Mechanism

doi: 10.3390/antiox11112110

Figure Lengend Snippet: Western Blot signal as a function of tubulin concentration. The range of protein concentration for measuring the XPB, XPA and XPD levels in BRBE-treated and untreated HeLa cells were performed within the linear protein concentration range shown in this graph.

Article Snippet: The blots were stained with XPA (sc-28353), XPB (sc-271500), anti -XPD (sc-101174), and anti -Tubulin (sc-53140) primary monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Concentration Assay, Protein Concentration

Journal: Molecular Cancer Research

Article Title: BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification

doi: 10.1158/1541-7786.MCR-22-0635

Figure Lengend Snippet: MeT5A BAP1 w-/KO cells recapitulate phenotypic aspects of BAP1-deficient MPM cells. A, Sequence confirmation of edited alleles: introduction of the w-family splice site mutation to induce exon 7 skipping (left) and a promoter trap to knockout (KO) BAP1 expression (right) in the isogenic MeT5A cells. B, BAP1 protein levels are constitutively reduced in BAP1 w-/+ and BAP1 w-/KO cells. Representative immunoblot and quantification relative to actin, mean of three independent experiments, error bars SD, one-way ANOVA with Dunnett post hoc test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. C, BAP1 deficiency slows proliferation of MeT5A cells. ATP-luciferase assay for BAP1 w-/KO C5.1; mean of three independent experiments, error bars SD, t test. **, P < 0.01 compared with MeT5A-BAP1 +/+ . D, BAP1 deficiency causes MeT5A cells to accumulate in S-phase. Cell-cycle distribution determined by flow cytometry for BAP1 +/+ and BAP1 w-/KO C5.1 (supporting data, Supplementary Fig. S5E). Mean of three independent experiments, error bars SD; unpaired t test; *, P = 0.0303; **, P = 0.0014. E, H2A ubiquitylation is increased in BAP1 -mutated MeT5A cells. Representative immunoblot and quantification, mean of three independent experiments, error bars SD, one-sample t test. **, P < 0.01.

Article Snippet: Primary antibodies were mouse anti-ASS1 (2C10 MABN704, Sigma-Aldrich; RRID:AB_2927674), BAP1 (C-4 sc-28383, Santa Cruz Biotechnology; RRID:AB_626723), PGK1 (22C5D8 ab113687, Abcam; RRID:AB_10861977), and β-actin (ab6276, Abcam; RRID:AB_2223210); rabbit anti-AK4 (HPA049461, Sigma-Aldrich; RRID:AB_2680776), DHFR (ab124814, Abcam; RRID:AB_10975115), ENO2 (#9536, Cell Signaling Technology; RRID:AB_2099308), GLS (ab156876, Abcam; RRID:AB_2721038), histone H2A (07-146, Upstate; RRID:AB_310394), H2A-Ub (#8240, Cell Signaling Technology; RRID:AB_10891618), MDH2 (HPA019716, Sigma-Aldrich; RRID:AB_1853680), OGT (#5368S, Cell Signaling Technology; RRID:AB_11217621), PGAM1 (ab96622, Abcam; RRID:AB_10687155), PGM2 (ab151746, Abcam; RRID:AB_2927675), PYGL (HPA004119, Sigma-Aldrich; RRID:AB_1079723), RRM1 (3388, Cell Signaling Technology; RRID:AB_2180382) and β-actin (A2066, Sigma-Aldrich; RRID:AB_476693), or goat anti-ALDOC (D-14 sc-12066, Santa Cruz Biotechnology; RRID:AB_2226594), IDH3B (ab118287, Abcam; RRID:AB_10899317), and SUCLG2 (A-15 sc-99646, Santa Cruz Biotechnology; RRID:AB_10843272).

Techniques: Sequencing, Mutagenesis, Knock-Out, Expressing, Western Blot, Luciferase, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells

doi: 10.3390/ijms231911649

Figure Lengend Snippet: Protein analysis of the three XPA mutated subclonal cell lines compared to XPA WT. ( A ) Predicted AA sequences of homozygous (WT, B1) and heterozygous (A9, A3) cell lines following the regular transcription and translation start sites are depicted. The XPA WT is based on NCBI transcript CCDS6729. AA sequences corresponding to the sgRNA-targeted regions are shown in green, altered AA sequences in red. Stars indicate translation termination sites upon the first in-frame stop codon. The approximate epitopes of the anti-XPA antibodies are highlighted for D9U5U (purple, targeting ≈ 50 AA surrounding the published target AA R158) and STJ96279 (blue) clones. The epitope of the 12F5 antibody is not published. ( B ) Immunoblotting with the 12F5 antibody showed complete loss of full length XPA in exon 1 knockout cell lines, B1 (p.A7Lfs*8) and A9 (p.A7Lfs*8/p.A7Gfs*55), while still producing a weaker XPA signal in A3 (p.I69_D70delinsN/p.D70Hfs*31) cells. Using D9U5U and STJ96279, a smaller band was newly detected in B1, presumably representing a de novo XPA variant. The AA one-letter-code was used in both panels. AA, amino acid(s); sgRNA, single-guide RNA; WT, wildtype.

Article Snippet: They were probed with antibodies against XPA with three different antibody clones: 12F5 (Santa Cruz Biotechnology, Dallas, TX, USA), D9U5U (Cell Signaling Technology, Danvers, MA, USA) and STJ96279 (St John’s Laboratory, London, UK). β-Actin (detected by antibody clone AC-15, Merck, Darmstadt, Germany) served as loading control.

Techniques: Clone Assay, Western Blot, Knock-Out, Variant Assay

Journal: Cancer Epidemiology Biomarkers & Prevention

Article Title: Expression of Nucleotide Excision Repair Proteins in Lymphocytes as a Marker of Susceptibility to Squamous Cell Carcinomas of the Head and Neck

doi: 10.1158/1055-9965.epi-05-0101

Figure Lengend Snippet: Figure 1. Reproducibility and linearity of reverse-protein microarray assay data. A. Serial dilutions of four cell-extract samples (1, 2, 3, and 4) were spotted in triplicate and probed with specific antibodies against XPA and XPF. Each slide was tested three times (repeats 1, 2, and 3). The statistical data are summarized in Table 1. B. The mean intensities of the spots in (A; in arbitrary units) were plotted on a log scale against the number of dilutions of the cell extract. C. Reverse-protein microarray assay of expression of the NER proteins ERCC1, XPA, XPC, XPD, XPF, and XPG. The level of h-actin was used as an internal control for standardization of the expression levels. The statistical data are summarized in Table 3.

Article Snippet: We used mouse anti-human monoclonal or anti-goat or anti-rabbit polyclonal antibodies against XPD and XPG (Santa Cruz Biotechnology, Santa Cruz, CA); XPA, XPC, and XPF (Abcam, Cambridge, MA); ERCC1 (Novus Biological, Littleton, CO); and h-actin (Sigma).

Techniques: Microarray, Expressing, Control