rabbit anti 53bp1  (Novus Biologicals)

Journal: Nature Communications

Article Title: HELZ is a RNA-DNA helicase that resolves R loops to facilitate homologous recombination repair

doi: 10.1038/s41467-026-75089-3

Figure Lengend Snippet: a , b Representative images and quantification for 53BP1 nuclear bodies in U2OS cells silenced for HELZ or a NT control and rescued for spontaneous 53BP1 nuclear bodies with overexpression of mCherry-RNaseH1. c U2OS cells transfected with indicated siRNA were seeded for colony formation. 72 h post-transfection, cells were treated with indicated doses of ABT888 every 2-3 days. Percent survival colonies are shown. Mean ± SD, n = 3 independent experiments. d HEK293T cells containing an integrated DR-GFP HR reporter were silenced with indicated siRNA and transfected with I-SceI endonuclease and RFP (−) or RFP-HELZ (WT, K674N) as shown. Live cells collected were subjected to flow cytometry. RFP-expressing cell population was sorted, and percent of GFP-positive cells within this population were determined to analyze HR efficiency. Mean ± SD, n = 3 independent experiments. e Western blot analysis showing HELZ knockdown and expression of RFP-HELZ WT and K674N from ( d ). f , g U2OS cells were depleted for HELZ for 72 h, treated with 4 Gy IR with or without RNase H or RNase A treatment in live cells for 1 h before fixation, and processed 4 h after IR for indirect immunofluorescence with indicated antibodies. Representative images and quantitation ( n = 7 independent experiments) are shown. The median is indicated by a horizontal line. h 293T cells were transfected with NT, HELZ, or BRCA1 siRNA and 72 h later, treated with 4 Gy IR and processed for western blot analysis with indicated antibodies. i , j U2OS cells were transfected with HELZ or NT siRNA for 72 h and mCherry-RNaseH1 WT or D10R/E48R for 48 h, treated with 4 Gy IR, and processed 4 h later for indirect immunofluorescence with indicated antibodies. Representative images and quantitation are shown ( n = 3 independent experiments). Statistical analyses used one-way ANOVA ( c ) or two-way ANOVA ( b , d , g , j ) followed by Dunnett’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns not significant. All medians are indicated by a horizontal line ( b , j , g ). Source data are provided as a file.

Article Snippet: Primary antibodies used for western blotting, immunoprecipitation (IP) and immunofluorescence (IF) are as follows: HELZ (ThermoFisher customized 1:50 for Western; Proteintech # 26635-1-AP; 1:1000 for Western, 1.2 mL per 1 mg DNA for ChIP, 1:1000 for PLA); S9.6 (Millipore #MABE1095; 1:600 for Slot Blot, 1:250 for IF, 0.5 mL per one million cell nuclei for DRIP-WB, 5U per 1 mg DNA for DRIP); ssDNA (Millipore #MAB3868; 1:1000 for Slot Blot); IgG (Invitrogen # 10500 C and Sigma # N103 for ChIP); GFP (Santa Cruz Tech # SC996; 1:1000 for Western; and abcam # ab290; 1:5000 for Western, 1 μg per 2 mg lysate for IP); γH2AX (Cell Signaling Technology # 2577S; 1:500 for IF; Millipore # 05-636; 1:6000 for IF; and Active Motif # 39117; 1 mL per 1 mg DNA for ChIP); KU80 (abcam # 80592; 1:1000 for Western); GAPDH (Santa Cruz tech # sc-47724; 1:1000 for Western); 53BP1 (Bethyl # A300-273A; 1:1000 for IF); RPA32 (Santa Cruz Tech # sc-14692; 1:400 for Western); pRPA32 S4/8 (Bethyl # A700-009; 1:1000 for Western); RPA70 (Cell Signaling # 2267S; 1:120 for IF); BRCA1 (Millipore # 07-434; 1:1000 for Western; 1.25ug/mg lysate for IP); CtIP (Millipore # MABE1060; 1:1000 for Western); Senataxin (Novus biological # NB100-57542; 1:1000 for IF) Rad51 (abcam # ab176458; 1:1000 for IF); Lamin A/C (Cell Signaling #2032; 1:1000 for Western) RFP (abcam # ab125244; 1:1000 for Western); α-Tubulin (Sigma # T6074; 1:10,000 for Western); and BrdU (BD Biosciences # 347580; 1:200 for IF).

Techniques: Control, Over Expression, Transfection, Flow Cytometry, Expressing, Western Blot, Knockdown, Immunofluorescence, Quantitation Assay, Comparison

Journal: bioRxiv

Article Title: FBH1 and RAD54L directly interact and cooperate to drive replication fork reversal

doi: 10.64898/2026.05.22.727240

Figure Lengend Snippet: ( A ) Schematic of the DNA fiber assay protocol used in (B). ( B ) Dot plot with medians of IdU/CldU ratios in HeLa RAD54L KO cells and RAD54L KO cells with wild type RAD54L or mutant RAD54L-BM ( i . e ., RAD54L-4A/S49E ) and transfected with negative control (Ctrl) or 53BP1 siRNA (n=2; 124-318 fiber tracts analyzed; ns=not significant, ****p < 0.0001). ( C ) Schematic of the DNA fiber assay protocol used in (D). ( D ) Dot plots with medians of IdU tract lengths in HU-treated and untreated U2OS FBH1 KO cells and FBH1 KO cells expressing wild-type FBH1 or E3 ligase-deficient FBH1 ( i . e ., FBH1-F266A/P267A ) and transfected with negative control (Ctrl) or RAD54L siRNA (n=2; 219-401 fiber tracts analyzed; ns=not significant, ****p < 0.0001; Kruskal-Wallis test followed by Dunn’s multiple comparisons test).

Article Snippet: The following primary antibodies were used: α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:1000), α-RAD51(Ab-1; EMD Millipore; 1:4000), α-FBH1 (sc-81563;1:2000), α-SMARCAL1 (sc-166209; 1:300), α-HA (16B12; 901533; BioLegend), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1000), α-GFP (50430-2-AP; Proteintech; 1:8,000), α-53BP1 (A300-272A; Bethyl Laboratories; 1:30,000).

Techniques: Mutagenesis, Transfection, Negative Control, Expressing