antibody against slx4  (Novus Biologicals)

Journal: Cancer Biology & Therapy

Article Title: SLX1 silencing overcomes Olaparib resistance in metastatic castration-resistant prostate cancer by disrupting SLX4-mediated DNA repair complexes

doi: 10.1080/15384047.2025.2545062

Figure Lengend Snippet: Knockdown of SLX1 reduces the binding of ERCC1-XPF, PLK1, and TOPBP1 to the SLX1-SLX4 complex.

Article Snippet: Equal amounts of protein (1000 μg) were incubated overnight at 4°C with 1 μg of the indicated primary antibody against SLX4 (NBP1–28680, Novus Biologicals).

Techniques: Knockdown, Binding Assay

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: SLX4 interacts with both MutSα and MutSβ through a conserved N-terminal region. ( A ) HeLa FITo cells were transfected with FLAG-HA-SLX4 (FHA-SLX4) vector before FLAG immunoprecipitation (IP) and western blotting, which shows that MSH2 co-immunoprecipitates with overexpressed SLX4. ( B ) IP of endogenous SLX4. Co-immunoprecipitation (coIP) of MSH2 with endogenous SLX4 is barely detectable compared to experiments using overexpressed FHA-SLX4 as in A. The asterisk indicates an aspecific band recognized by the anti-SLX4 antibody ( C ) Left panel: IP of endogenous SLX4 from a nuclear soluble fraction or a chromatin-solubilized fraction in which the coIP of MSH2, MSH3 and MSH6 is readily detected. Right panel: control of the fractionation by western blot against CHK1, mainly nuclear soluble, and SMC3 enriched in chromatin. ( D ) Scheme of SLX4 illustrating the location and conservation of a short domain representing the putative MSH2 binding domain (MSH2bd) deleted in SLX4 ΔMSH2bd . Alignments were performed with ProViz . ( E ) HeLa FITo cells were transfected with FHA-SLX4 WT, FHA-SLX4 ΔMSH2bd or FHA-SLX4 FLW* , which is deficient for XPF binding, before FLAG IP and western blotting with the indicated antibodies.

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Fractionation, Binding Assay

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: Characterization of the KO30 cell line expressing an N-terminally truncated form of SLX4. ( A ) Western blot showing that HeLa FITo KO1 and KO30 clones generated by CRISPR-Cas9 express a truncated form of SLX4 termed SLX4ΔNter (indicated by an arrow), the expression of which is sensitive to a siRNA that targets SLX4 mRNA. ( B ) SLX4ΔNter protein starts at Methionine 360; see supplementary results for details. ( C ) Clonogenic survival assay in response to mitomycin C (MMC) of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or UBZ-mutated (UBZmut). Cells were treated for 24 h with the indicated dose of MMC ( n = 2–4 experiments, mean ± SD are represented).

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Expressing, Western Blot, Clone Assay, Generated, CRISPR, Clonogenic Cell Survival Assay

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: Interaction of MSH2 and SLX4 is not required for ICL repair. ( A ) Clonogenic survival assay in response to MMC of HeLa FITo cells transfected with control siRNA (siLUC) or siRNA targeting MSH2 (siMSH2) ( n = 3 for MMC 2 ng/ml, n = 4 for MMC 5 ng/ml, mean ± SEM are represented on the graph). ( B ) Complementation of KO30 cells with FHA-SLX4 WT or SLX4 ΔMSH2bd . Induction of exogenous SLX4 expression was achieved with 2 ng/ml of doxycycline as in (C) and (D). ( C ) Clonogenic survival assay of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd in response to MMC ( n = 3 for MMC 1 ng/ml, n = 5 or 6 for MMC 2 ng/ml, mean ± SD are represented). ( D ) Same as in (C) except that Melphalan (500 nM) was used as an alternative crosslinking agent ( n = 3, mean ± SD are indicated).

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Clonogenic Cell Survival Assay, Transfection, Control, Expressing

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: Interaction of SLX4 and MSH2 contributes to the toxicity of 6-thioguanine (6-TG) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). ( A ) Clonogenic survival assay of HeLa FITo cells transfected with the indicated siRNA in response to a 24 h treatment with 6-TG ( n = 4–7 experiments, mean ± SEM are represented). ( B ) Clonogenic survival assay in response to 6-TG of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd ( n = 3–5 experiments, mean ± SEM are represented). ( C ) Clonogenic survival assay in response to MNNG of KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd ( n = 3–4 experiments, mean ± SEM are represented). ( D ) KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd were treated with MNNG (0.1 μM) for 24 h before drug removal and addition of fresh medium. Cells were collected at the indicated time points of recovery (+rec) and induction of the DNA damage response was analysed by western blot.

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Clonogenic Cell Survival Assay, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: The MSH2 binding domain of SLX4 is a SHIP box that inhibits mismatch repair and antagonizes EXO1–MSH2 interaction. ( A ) Weblogo representation of multiple sequence alignments of SHIP boxes of Exo1 and Fun30 from fungal species in the Saccharomycotina ( , ). ( B ) HeLa FITo cells were transfected with expression vectors coding for GFP, FLAG-SLX4, GFP-MSH2 WT and/or GFP-MSH2 M453I , as indicated, before GFP pull-down and western blotting with the indicated antibodies. The asterisk represents the remaining signal for EXO1 when blotting for MSH3 after prior blotting for EXO1. ( C ) Peptide pull-down using biotinylated SLX4 peptides that contain a WT or mutated SHIP box, immobilized on Streptavidin-coated beads and incubated with HeLa nuclear extracts. Residues selected for mutagenesis are shown in bold in the WT sequence. ( D ) In vitro mismatch repair assay using a plasmid containing a G/T mismatch and a 5′ nick incubated with nuclear extracts (NE) from HeLa FITo cells as described in Material and Methods. Where indicated, WT or mutant SHIP peptides (80.5 μM) were added to the reaction. As a negative control, the NE was inactivated at 95°C before the reaction. DNA was purified and digested with ApalI and PvuII. Repair of the mismatch restores the PvuII site and produces two bands of 1.55 and 1.03 kb on an agarose gel. The percentage of repair is indicated.

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Binding Assay, Sequencing, Transfection, Expressing, Western Blot, Incubation, Mutagenesis, In Vitro, Plasmid Preparation, Negative Control, Purification, Agarose Gel Electrophoresis