ascc2  (Proteintech)

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 recruits ASCC3 to stalled forks in a manner dependent upon ASCC2’s ubiquitin binding activity. ( A ) Western blot analyses of U2OS WT, ASCC2-KO, and ASCC3-KO cells. Immunoblotting was performed with anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. The γ-tubulin blot was used as a loading control in this and subsequent figures except for where the α-tubulin blot was used as a loading control. The asterisk (*) indicates a non-specific band. ( B ) Representative images of endogenous ASCC2-EdU PLA foci formation in U2OS WT and ASCC2-KO that were either treated without or with 4 mM HU for 4 h. Nuclei were stained with DAPI in blue in this and subsequent figures. Scale bars in this and subsequent figures: 5 μm. ( C ) Quantification of endogenous ASCC2-EdU PLA foci formation from (B). The PLA experiments were performed independently twice with reproducible data in this, 1F, 1G, and 1H panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 492-506 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( D ) Quantification of PLA foci formation in U2OS treated with 4 mM HU for 4 h. PLA assays were performed in several conditions as indicated. This PLA experiment was performed once. A total of 492-515 cells per condition were analyzed. **** P <0.0001. ( E ) Representative images of endogenous ASCC3-EdU PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( F ) Quantification of endogenous ASCC3-EdU PLA foci formation from (E). A total of 1389-1396 cells per condition were analyzed. **** P <0.0001. ( G ) Quantification of Myc-tagged ASCC2-EdU PLA foci formation in no HU- or HU-treated U2OS ASCC2-KO cells expressing various Myc-ASCC2 alleles as indicated. A total of 603-617 cells per condition were analyzed. **** P <0.0001. ( H ) Quantification of endogenous ASCC3-EdU PLA foci formation in no HU- or HU-treated U2OS ASCC2-KO cells stably expressing various Myc-ASCC2 alleles as indicated. A total of 806-816 cells per condition were analyzed. **** P <0.0001. ( I ) Western blot analyses of U2OS ASCC2-KO cells stably expressing various Myc-ASCC2 alleles as indicated. Immunoblotting was performed with anti-ASCC3 and anti-γ-tubulin antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Ubiquitin Proteomics, Binding Assay, Activity Assay, Western Blot, Control, Staining, MANN-WHITNEY, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 interacts with polyubiquitylated PCNA and is recruited by PCNA ubiquitylated at K164 to stalled forks. ( A ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS cells transfected with siControl or siRNA against SHPRH, HLTF or RFWD3. The PLA experiments were performed twice independently with reproducible data in this, 2D, and 2E panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 805-846 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( B ) Western blot analyses of U2OS WT and SHPRH-KO cells. Immunoblotting was performed with anti-SHPRH, anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. ( C ) Representative images of ASCC2-EdU PLA foci in HU-treated U2OS WT and SHPRH-KO cells. ( D ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS WT and SHPRH-KO cells from (C). U2OS ASCC2-KO cells were included as a control. A total of 817-822 cells per condition were analyzed. **** P <0.0001. ( E ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS SHPRH-KO cells expressing various Myc-SHPRH alleles as indicated. A total of 827-832 cells per condition were analyzed. **** P <0.001. ( F ) Representative images of ASCC2-EdU PLA foci in no HU- or HU-treated RPE1 parental (WT), A1 (PCNA-K164R), and B1 (PCNA-K164R) cells. ( G ) Quantification of ASCC2-EdU PLA foci formation in RPE1 WT, A1, B1, A1 revertant (A1.R, PCNA-K164K), and B1 revertant (B1.R, K164K) cells that were treated with or without HU. This PLA experiment was done once. A total of 525-540 cells per condition were analyzed. **** P <0.0001. ( H ) Coimmunoprecipitation with anti-HA antibody in UV-irradiated, ATRi-treated and USP1-depleted HEK293T cells expressing the vector alone, HA-tagged ASCC2, or HA-tagged ASCC2 carrying LLL-AAA mutations. Immunoblotting was performed with anti-Ubiquityl-PCNA (K164), anti-PCNA, and anti-HA antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, MANN-WHITNEY, Western Blot, Control, Expressing, Irradiation, Plasmid Preparation

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 interacts with RPA and this interaction is further induced by replication stress. ( A ) Anti-Myc coIPs in HEK293T cells transfected with eGFP-ASCC3 together with either the vector alone, Myc-RPA32, or Myc-RPA70. Immunoblotting was done with anti-Myc and anti-GFP antibodies. ( B ) Anti-Myc coIPs in HEK293T cells transfected with HA-RPA70 together with either the vector alone or Myc-ASCC3. Immunoblotting was done with anti-Myc and anti-HA antibodies. ( C ) Anti-Myc coIPs in HEK293T cells transfected with HA-RPA32 together with the vector alone or Myc-ASCC3. Immunoblotting was done with anti-Myc and anti-HA antibodies. ( D ) Coimmunoprecipitations (coIPs) with IgG, anti-ASCC2, or anti-ASCC3 antibodies in HCT116 cells. Immunoblotting was done with anti-ASCC2, anti-ASCC3, anti-RPA70, and anti-RPA32 antibodies. ( E ) Anti-RPA32 coIPs in HCT116 cells. IgG was used as a control. Immunoblotting was performed with anti-RPA32 and anti-ASCC3 antibodies. ( F ) Anti-HA coIPs in no HU- or HU-treated HEK293T cells expressing the vector alone or HA-RPA70. Immunoblotting was done with anti-HA, anti-ASCC2, and anti-ASCC3 antibodies. ( G ) Anti-HA coIPs in HEK293T cells expressing the vector alone or HA-RPA70 in the presence or absence of DNase I. Immunoblotting was done with anti-HA and anti-ASCC3 antibodies. ( H ) Representative images of ASCC3-RPA32 PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( I ) Quantification of ASCC3-RPA32 PLA foci formation from (H). The PLA experiments were performed twice independently with reproducible data. Data from one representative experiment are shown as scatter plot graphs with the mean indicated. A total of 402-418 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test. **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing, MANN-WHITNEY