HLTF (E9H5I) Rabbit mAb 45965S from Cell Signaling Technology

Published Figures

Register or Sign In to View Figures.

Nucleic Acids Research
November 11 2024

Fig 1: RAD54L’s branch migration activity is specifically required for the FBH1 pathway of RAD51-mediated fork reversal. (A) Schematic of the DNA fiber assay protocol used in (C). (B) Representative Western blots to show extent of FBH1 and HLTF knockdown in RAD54L KO cells (lanes 4–6) and RAD54L KO cells expressing wild type RAD54L (lanes 1–3) or mutant RAD54L-4A/S49E (lanes 7–12). RAD54L-4A/S49E-1 dignifies a clonal isolate; RAD54L-4A/S49E #2 dignifies a puromycin-resistant cell population. Loading control: α-tubulin. (C) Dot plot with medians of IdU fork speeds in HU-treated RAD54L KO cells and RAD54L KO cells expressing wild type RAD54L or RAD54L-4A/S49E and transfected with Ctrl, FBH1 or HLTF siRNA (n = 3; 85–224 fiber tracts/experiment analyzed; n = 1 for RAD54L-4A/S49E #2). (D) Schematic of the DNA fiber assay protocol used in (F). (E) Representative western blots to show extent of BRCA2 knockdown in RAD54L KO cells (lanes 3–4) and RAD54L KO cells expressing wild type RAD54L (lanes 1–2) or mutant RAD54L-4A/S49E (lanes 5–8) and RAD54L-K189R (lanes 9–10). RAD54L-4A/S49E-1 dignifies a clonal isolate; RAD54L-4A/S49E #2 and RAD54L-K189R #1 dignifies puromycin-resistant cell populations. Loading control: MSH2. (F) Dot plot with medians of IdU/CldU tract length ratios in HU-treated RAD54L KO cells and RAD54L KO cells expressing wild type RAD54L or mutant RAD54L-4A/S49E and RAD54L-K189R transfected with Ctrl or BRCA2 siRNA (n = 3; 46–104 fiber tracts/experiment analyzed; n = 1 for RAD54L-K189R #1). Data were analyzed by Kruskal–Wallis test followed by Dunn's multiple comparisons test (ns, not significant; ∗∗∗∗P< 0.0001). (G) Model to explain the role of RAD54L in the FBH1 pathway of RAD51-mediated fork reversal. RAD54L may be recruited through its interaction with RAD51 on extended parental ssDNA. FBH1 is recruited to stalled replication forks by PCNA ((78); PCNA is not shown here). Unwinding of lagging strand DNA through FBH1 (72) may initiate nascent strand annealing. RAD54L may then oligomerize on a 4-way junction and drive reversal through BM. The dependency of FBH1 on RAD54L may be indicative of coordinated recruitment and/or post-translational modification orchestrating sequential coaction. The number of subunits in the RAD54L BM oligomer remain to be determined. Schematic created with Biorender.com.
Register or Sign In to View Figures.

Nucleic Acids Research
November 11 2024

Fig 2: Loss of RAD54L does not hinder replication restart, leads to faster replication during mild replication stress, and partially restores fork restraint in cells with FBH1 knockdown. (A) Schematic of the DNA fiber assay protocol used to assess replication restart. (B) Dot plot with medians of IdU fork speeds in untreated (NT) or HU-treated parental Hs578T, RAD51AP1 KO, and RAD54L KO cells (n = 3; 65–113 fiber tracts/experiment analyzed). (C) Fractions of stalled and restarted forks after 4 mM HU in the Hs578T cells and derivatives shown in (B). (D) Dot plot with medians of CldU fork speeds in untreated (NT) or HU-treated Hs578T cells and derivatives (n = 3; 65–80 fiber tracts/experiment analyzed). (E) Schematic of the DNA fiber assay protocol used to evaluate the progression of replication forks during mild replication stress in (F). (F) Dot plot with medians of IdU fork speeds in parental HeLa cells, RAD54L KO cells expressing RAD54L KO, and two independently isolated RAD54L KO cell lines treated with or without 25 μM HU during the IdU pulse (n = 3; 82–220 fiber tracts/experiment analyzed). (G) Schematic of the DNA fiber assay protocol used to evaluate the progression of replication forks during mild replication stress in (H), (I) and (J). (H) Dot plot with medians of IdU fork speeds in hTERT RPE-1 cells transfected with Ctrl or RAD54L siRNA and treated with or without 25 μM HU during the IdU pulse (n = 3; 76–293 fiber tracts/experiment analyzed). (I) Dot plot with medians of IdU fork speeds in HeLa cells transfected with control (Ctrl), RAD54L, FBH1 or HLTF siRNA and treated with or without 25 μM HU during the IdU pulse (n = 3; 92–139 fiber tracts/experiment analyzed). (J) Dot plot with medians of IdU fork speeds in HeLa and RAD54L KO cells transfected with Ctrl, FBH1, HLTF or SMARCAL1 siRNA (n = 3; 122–420 fiber tracts/experiment analyzed). All data were analyzed by Kruskal–Wallis test followed by Dunn's multiple comparisons test (ns, not significant; ∗P< 0.05; ∗∗∗∗P< 0.0001; or as indicated).

Be the first to write a review!

Citations:

(3)

Figures:

$339.00 Inquire

from
Cell Signaling Technology for
HLTF (E9H5I) Rabbit mAb

Cell Signaling Technology's HLTF (E9H5I) Rabbit mAb is a Rabbit monoclonal antibody. The HLTF (E9H5I) Rabbit mAb Antibody was generated using HLTF as the antigen. It reacts with Homo Sapiens, Human, Mouse, Rat, and Non-Human Primate. This antibody has been shown to work in applications such as: Precipitation, Immunoprecipitation, and Western Blot.

Nucleic Acids Research

Nucleic Acids Research

HLTF (E9H5I) Rabbit mAb from Cell Signaling Technology

Cell Signaling Technology

Citations (3)

Reviews

HLTF (E9H5I) Rabbit mAb from Cell Signaling Technology

About Biocompare

Advertise with Us

Specialized Search Tools

Site Map