antibody against fancd2 nb100-182ss  (Novus Biologicals)

Journal: Nature Communications

Article Title: ANP32E drives vulnerability to ATR inhibitors by inducing R-loops-dependent transcription replication conflicts in triple negative breast cancer

doi: 10.1038/s41467-025-59804-0

Figure Lengend Snippet: a Dot blot showing R-loop abundance in tIMEC, tIMEC-A, and tIMEC-A-H1 cells ± RNaseH1. dsDNA used as internal normalizer. Quantification from three biological replicates (mean ± s.e.m.), unpaired two-tailed t -test p values shown. b Representative PLA images showing proximity between ANP32E and R-loops. Scale bar = 10 µm. c PLA images of pS2RNApol II-PCNA proximity as TRCs marker. Scale bar = 10 µm. d Quantification of PLA foci/nucleus from ( b , c ): tIMEC n = 106/104, tIMEC-A n = 85/84, tIMEC-A-H1 n = 72/75. Unpaired two-tailed t -test p values are shown. e Top: FANCD2 immunofluorescence in EdU+ cells (EdU channel excluded for clarity). Bottom: quantification of FANCD2 foci/nucleus in EdU+ (green violins) and EdU– (gray violins) cells; unpaired two-tailed t -test p values shown. Scale bar = 10 µm. f Alkaline comet assay ± VE822 (1.25 µM, 24 h). Top: representative images, comet quantification (OpenComet) , and DNA intensity heatmaps (CometScore) . Bottom: violin plots of comet tail length; 150–300 cells/condition, three biological replicates. Unpaired two-tailed t -test was used for p value calculation. g Left: representative binucleated (BN) cells with micronuclei (MNi) after Cytochalasin B + Mitomycin C treatment. Right: %BN cells with MNi ± Mitomycin C (0.03 µg/ml) or VE822 (0.027, 0.08, 0.25 µM) treatment. ≥3000 cells/condition on three biological replicates. The barplot reports mean ± s.d. with two-way ANOVA adjusted p values. h Immunostaining of BN cells with MNi that are positive or negative for centromere staining (CREST) after VE822. Phalloidin marks cytoplasm; arrows indicate MNi. Scale bar = 10 µm. i Barplot showing the percentage of BN cells with DSB-derived (acentric) MNi ± Mitomycin C or VE822 treatment. Mean ± s.e.m. from three replicates; 25–60 BN cells/condition were quantified per replicate.

Article Snippet: Primary antibodies against FANCD2 1:100 (NovusBio, NB100-182SS), pRPA32 1:1000 (Bethyl, A300-246A-8), and 53BP1 1:100 (Millipore, MAB3802) were diluted in blocking solution and incubated at RT for 2 h. Next, coverslips were washed in PBS 1x before incubation with Alexa-Fluor-647 specie-specific secondary antibodies (Thermo Fisher, A32728 and A-21245) and either Hoechst 1:2000 for EdU labeled cells or DAPI 1:1000 for simple IF.

Techniques: Dot Blot, Two Tailed Test, Marker, Immunofluorescence, Alkaline Single Cell Gel Electrophoresis, Immunostaining, Staining, Derivative Assay

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 6. CDC73-mediated activation of CDK12/13 is essential for balanced gene expression (A) Volcano plot of significance and difference of spike-in and library normalized gene expression across cell lines indicated relative to siControl sample. Significantly upregulated (log2FC > 1 and adjusted p value < 0.05) and downregulated (log2FC < −1 and adjusted p value < 0.05) genes indicated in red and blue, respectively. The number of differentially expressed events are similarly indicated. Log2FC, log2 fold change. (B) Boxplot of log2FC of library and spike-in normalized nascent RNA read counts relative to the siControl condition, across gene intervals divided according to length. Long: > ∼100 kb, mid2: ∼30–100 kb, mid: ∼10–30 kb, short: < ∼1–10 kb. ****p < 0.0001, *p < 0.1, and ns: not significant as determined using an unpaired t test. (C) Metagene analysis of non-overlapping coding genes between 1 and 10 kb (n = 2,442) using library- and spike-in normalized nascent RNA read counts obtained from transient transcriptome chem sequencing (TTchem-seq). Line indicates mean signal of experiment performed in technical triplicate. Shaded area indicates standard error. Si73, siRNA targeting CDC73; CPM, counts per million; TSS, transcription start site; TES, transcription end site; kb, kilobases. (D) Representative images of nascent RNA read counts across GADD45B and HSPA1A, with the direction of transcription indicated by arrow. (E) Western blot of whole cell extracts of Flp-In T-Rex HEK293 cell lines with or without knockdown of CDC73 and induction of recombinant FLAG-CDC73 siRNA resistant WT and KIM mutant forms. ATR and FANCD2 blotted to show the loss of protein expression in the siCDC73 and the KIM mutants.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Activation Assay, Gene Expression, Sequencing, Western Blot, Knockdown, Recombinant, Mutagenesis, Expressing