nb100 618 rat anti primpol  (Novus Biologicals)

Journal: bioRxiv

Article Title: PRIMPOL promotes replication fork progression but not double strand break formation in FBH1-deficient cells in response to hydroxyurea

doi: 10.1101/2025.07.08.663736

Figure Lengend Snippet: (A) Western blot depicting FBH1 and PRIMPOL protein levels in U2OS cells. Vinculin is the loading control. (B) Schematic of the replication fiber experiment. (C) Dot plot depicting IdU tract lengths in U2OS cells after the inducted treatments. At least 200 replication tracts per condition. Red line indicates sample mean. (D) Western blot depicting FBH1 and PRIMPOL protein levels in RPE-1 cells. Vinculin is the loading control. (E) Dot plot depicting IdU tract lengths in RPE-1 cells after the inducted treatments. At least 200 replication tracts per condition. Red line indicates sample mean. n=3, **** p <0.0001 ANOVA; Tukey HSD

Article Snippet: The membrane was stained with primary antibodies recognizing FBH1(purification described above), PRIMPOL (kind Gift from Juan Mendéz ( )), and Vinculin (Cell signaling, 13901S, 1:1000) overnight at 4°C.

Techniques: Western Blot, Control

Journal: Nucleic Acids Research

Article Title: CAF-1 promotes efficient PrimPol recruitment to nascent DNA for single-stranded DNA gap formation

doi: 10.1093/nar/gkae1068

Figure Lengend Snippet: CHAF1A depletion is epistatic with PrimPol depletion and results in a decrease in PrimPol localization to replication forks. ( A and B ) BrdU alkaline comet assay showing that loss of PrimPol is epistatic with loss of either CHAF1A or ASF1A in (A) BRCA2 KO or (B) WT HeLa cells. Accumulation of ssDNA gaps in samples depleted of either CHAF1A or ASF1A show no further decrease in ssDNA gaps when PrimPol is also depleted. At least 49 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown. ( C ) S1 Nuclease DNA fiber combing assay showing that CHAF1A co-depletion with PrimPol in WT or BRCA2KO HeLa cells does not result in a further decrease in ssDNA gap accumulation upon treatment with 0.4 mM HU. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). A schematic representation of the assay conditions is displayed. ( D ) BrdU alkaline comet assay showing that loss of PrimPol is epistatic with loss of either CHAF1A or ASF1A in MDA-MB-436 cells. Accumulation of ssDNA gaps in samples depleted of either CHAF1A or ASF1A show no further decrease in ssDNA gaps when PrimPol is also depleted. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is displayed. ( E ) S1 Nuclease DNA fiber combing assay showing that CHAF1A or ASF1A co-depletion with PrimPol in MDA-MB-436 cells does not result in a further decrease in ssDNA gap accumulation upon treatment with 0.4 mM HU. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). A schematic representation of the assay conditions is displayed. ( F and G ) BrdU alkaline comets showing the overexpression of PrimPol in BRCA2 KO HeLa cells under 0.4 mM HU (F) and 150 μM cisplatin (G) treatment is able to partially rescue ssDNA gap abundance when CHAF1A is knocked down. At least 50 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are displayed. ( H ) S1 Nuclease DNA fiber combing assay showing that overexpression of PrimPol in BRCA2 KO HeLa cells is not able to rescue ssDNA gap accumulation to an observable level upon treatment with 0.4 mM HU. At least 100 fibers were quantified per condition. The ratio of CldU to IdU tract lengths is presented with median values marked. Asterisks indicate statistical significance (Mann–Whitney test, two-tailed). A schematic representation of the assay conditions is displayed. ( I and J ) SIRF experiment showing that the increase in PrimPol localization to nascent DNA upon 0.4 mM HU treatment in BRCA2 KO HeLa cells is lost when either CHAF1A or ASF1A are depleted (I). Representative micrographs with scale bars representing 10 μm are shown (J). At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent SEM, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). A schematic representation of the assay conditions is shown.

Article Snippet: Antibodies used for western blot at 1:500 (unless otherwise noted) were: CHAF1A (Cell Signaling Technology 5480s); CHK1 (Cell Signaling Technology 2360s); FEN1 (Santa Cruz Biotechnology sc-28355); RPA2 (Abcam AB2175); RPA3 (Santa Cruz Biotechnology sc-271564); DNA2 (Abcam AB96488); ASF1A (Santa Cruz Biotechnology sc-53171); BRCA1 (Santa Cruz Biotechnology sc-6954); BRCA2 (Calbiochem OP95); GAPDH (Santa Cruz Biotechnology sc-47724); PrimPol (Proteintech 29824–1-AP) (1:250); Vinculin (Santa Cruz Biotechnology sc-25336).

Techniques: Alkaline Single Cell Gel Electrophoresis, MANN-WHITNEY, Two Tailed Test, Over Expression

Journal: Nucleic Acids Research

Article Title: CAF-1 promotes efficient PrimPol recruitment to nascent DNA for single-stranded DNA gap formation

doi: 10.1093/nar/gkae1068

Figure Lengend Snippet: Loss of ASF1A results in chemoresistance in BRCA-deficient cells. ( A ) Neutral comet assay showing that DSBs in BRCA2 KO HeLa cells induced by 150 μM cisplatin treatment for 2 h are suppressed with knockdown of ASF1A similarly to knockdown of CHAF1A. At least 47 nuclei were quantified per condition with median values marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are displayed. ( B ) γH2AX immunofluorescence showing that γH2AX foci induced by 150 μM cisplatin treatment for 2 h in BRCA2 KO HeLa cells are suppressed by knockdown of ASF1A similarly to knockdown of CHAF1A. At least 100 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance ( t -test two-tailed, unpaired). ( C ) Clonogenic survival assay showing that loss of ASF1A promotes cisplatin resistance in BRCA2 KO HeLa cells. The average of three experiments, with SEM indicated as error bars, is shown. Asterisks indicate statistical significance (two-way ANOVA). ( D ) Table summarizing the observed consequences of CHAF1A or ASF1A loss in BRCA-deficient cells on FP, RGS, DSB accumulation and drug response status. ( E ) Schematic model of the proposed model of CAF-1 role in efficient PrimPol recruitment to the replication fork and ssDNA gap generation. Created in BioRender. Moldovan, G. (2024) BioRender.com/y25 × 850.

Article Snippet: Antibodies used for western blot at 1:500 (unless otherwise noted) were: CHAF1A (Cell Signaling Technology 5480s); CHK1 (Cell Signaling Technology 2360s); FEN1 (Santa Cruz Biotechnology sc-28355); RPA2 (Abcam AB2175); RPA3 (Santa Cruz Biotechnology sc-271564); DNA2 (Abcam AB96488); ASF1A (Santa Cruz Biotechnology sc-53171); BRCA1 (Santa Cruz Biotechnology sc-6954); BRCA2 (Calbiochem OP95); GAPDH (Santa Cruz Biotechnology sc-47724); PrimPol (Proteintech 29824–1-AP) (1:250); Vinculin (Santa Cruz Biotechnology sc-25336).

Techniques: Neutral Comet Assay, Knockdown, MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Clonogenic Cell Survival Assay