Journal: Cell reports

Article Title: Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

doi: 10.1016/j.celrep.2023.112807

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Cdc37 , StressMarq Biosciences , Cat# SPC-142; RRID:AB_2570605.

Techniques: Virus, Recombinant, Proximity Ligation Assay, Software

Journal: Nucleic acids research

Article Title: PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

doi: 10.1093/nar/gkac948

Figure Lengend Snippet: Figure 2. Proteins recruited to persistently stalled replication forks. (A) Volcano plot displaying the results from n = 2 biologically independent SILAC iPOND experiments after 18 h hydroxyurea (HU) treatment against untreated control. FDR was calculated using limma. Selected proteins with an FDR < 5% are highlighted. (B) GO term analysis (Molecular Function, Biological Process and Cellular Compartment) of proteins enriched at persistently stalled replication forks (18 h HU) with an FDR < 5%. P-values were calculated by Fisher´s exact test and corrected for multiple comparisons using Benjamini–Hochberg correction. (C) Western blot validation of indicated proteins enriched by iPOND after 2 or 18 h hydroxyurea (HU) treatment. (D) Representative box plot of a proximity ligation assay (PLA) of n = 2 biologically independent experiments between TPX2 and RPA2 after treatment with DMSO or hydroxyurea (HU) for 2 or 18 h. Individual measurements of the mean PLA intensity per nucleus are plotted and the median indicated in red. P-values (****P-value < 0.0001) were derived from using one-way-ANOVA with Tukey correction for multiple comparisons. (E) Representative box plot of a proximity ligation assay (PLA) of n = 2 biologically independent experiments between PARP1 and RPA2 after treatment with DMSO or hydroxyurea (HU) for 2 or 18 h. Individual measurements of the mean PLA intensity per nucleus are plotted and the median indicated in red. P-values (****P-value < 0.0001) were derived from using one-way-ANOVA with Tukey correction for multiple comparisons.

Article Snippet: In vitro pull downs GFP-tagged TPX2 mutants were immobilized on preequilibrated GFP-Trap beads (Chromotek) in Co-IP buffer (20 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.2% glycerol, 0.2% Triton X-100, and protease inhibitor cocktail) for 1 h at 4◦C on a rotating wheel.

Techniques: Multiplex sample analysis, Control, Western Blot, Biomarker Discovery, Proximity Ligation Assay, Derivative Assay

Journal: Nucleic acids research

Article Title: PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

doi: 10.1093/nar/gkac948

Figure Lengend Snippet: Figure 4. TPX2 interacts with PARP1 and DNA repair factors (A) Volcano plot displaying n = 3 biologically independent experiments of LFQ-based interactome of a GFP-tagged TPX2 against a GFP control. The P-values were calculated by a two-sample t-test and adjusted for multiple comparisons using the Benjamini-Hochberg correction. Dotted lines indicate fold change >2, <–2 and 5% FDR. (B) Relative stoichiometry of the LFQ-based TPX2 interactions. Intensity-based absolute quantification (IBAQ) values for each interactor were normalized to the control IBAQ value. Data are represented as mean ± standard deviation. (C) Reactome pathways of TPX2 interacting proteins with log2 fold change >2 and FDR <5%. Terms are sorted by their adjusted P-value calculated using two-sided Fisher´s exact test with Benjamini–Hochberg correction. (D) Proximity ligation assay (PLA) between endogenous PARP1 and TPX2. The grey dots represent the individual values of the mean PLA intensity per nucleus; the red line indicates the median. P-values (****P-value < 0.0001) were derived using one-way-ANOVA with Tukey correction for multiple comparisons. (E) Western blot validation of indicated TPX2 interactors using an antibody against endogenous TPX2 or an IgG control for immunoprecipitation. (F) Schematics of full-length TPX2 and mutants used for pull down experiments. Numbers indicate the respective amino acid positions. Dark shaded regions indicate interaction domain with Aurora A and Importin. (G) Western blot analysis of an in vitro pull down between recombinant PARP1 and His-GFP-tagged TPX2 full-length protein and its mutants. Blue dots indicate presence of a protein in the respective condition.

Article Snippet: In vitro pull downs GFP-tagged TPX2 mutants were immobilized on preequilibrated GFP-Trap beads (Chromotek) in Co-IP buffer (20 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.2% glycerol, 0.2% Triton X-100, and protease inhibitor cocktail) for 1 h at 4◦C on a rotating wheel.

Techniques: Control, Quantitative Proteomics, Standard Deviation, Proximity Ligation Assay, Derivative Assay, Western Blot, Biomarker Discovery, Immunoprecipitation, In Vitro, Recombinant

Journal: Nucleic acids research

Article Title: PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

doi: 10.1093/nar/gkac948

Figure Lengend Snippet: Figure 5. TPX2 modulates PARP1 activity in vitro and in vivo. (A) Representative boxplot of n = 2 biologically independent experiments displaying the mean ADP-ribosylation per nucleus after a TPX2 (blue) or a control knockdown (grey) for 24 h. Cells were treated with 2 mM H2O2 for 10 min ± addition of 1 M Olaparib and ±1 h recovery. The centres of the boxplots indicate the median, limits the 25th–75th percentile, whiskers the 10th–90th percentile, and dots indicate outliers. ****P-value < 0.0001, one-way ANOVA with Tukey correction for multiple comparisons. (B) ADP-ribosylation western blot after TPX2 knockdown in U2OS cells with three individual siRNAs after 2 mM H2O2 for 10 min. Control cells were pre-treated for 1 h with 1 M olaparib. (C) Representative boxplot of n = 2 biologically independent experiments displaying the mean ADP-ribosylation per nucleus after a TPX2 (blue) or a non-targeting control knockdown (grey) for 24 h. Cells were treated with 10 mM PARGi for 1 h ± addition of 1 M Olaparib. The centres of the boxplots indicate the median, limits the 25th–75th percentile, whiskers the 10th–90th percentile, and dots indicate outliers. ****P-value < 0.0001, one- way ANOVA with Tukey correction for multiple comparisons. (D) Western blot displaying endogenous auto-ADP-ribosylation of PARP1 after TPX2 or control knockdown. (E) Representative Western blot of n = 2 biologically independent experiments of the in vitro ADP-ribosylation assay with purified PARP1 and TPX2 proteins (full-length and N mutant). Staining with an antibody against poly-ADP-ribosylation (left) and Ponceau staining (right). (F) Representative images of a proximity ligation assay (PLA) between endogenous HPF1 and PARP1 after knockdown of TPX2 or a control knockdown. PLA signal is displayed in red; Hoechst33342 staining is shown in blue. (G) Quantification of a PLA between HPF1 and PARP1 after TPX2 (turquoise) or control knockdown (grey). Individual values of the mean PLA intensity per nucleus are shown; the red line indicates the median. Antibody leave-out controls are shown in black. ****P-value < 0.0001, one-way ANOVA with Tukey correction for multiple comparisons. (H) Representative Western blot of the co-immunoprecipitation (IP) of GFP-tagged TPX2 after either a control knockdown or HPF1 knockdown. Inputs are shown on the left, GFP-IP on the right. * indicates the residual PARP1 staining after re-probing with the GFP antibody, while **indicates the specific HPF1 band. (I) Bar plot showing the mean ± SD derived from n = 2 biologically independent experiments of the GFP-TPX2 co-IP after HPF1 (blue) or control knockdown (grey). *P-value < 0.05, student´s t-test.

Article Snippet: In vitro pull downs GFP-tagged TPX2 mutants were immobilized on preequilibrated GFP-Trap beads (Chromotek) in Co-IP buffer (20 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.2% glycerol, 0.2% Triton X-100, and protease inhibitor cocktail) for 1 h at 4◦C on a rotating wheel.

Techniques: Activity Assay, In Vitro, In Vivo, Control, Knockdown, Western Blot, Purification, Mutagenesis, Staining, Proximity Ligation Assay, Immunoprecipitation, Derivative Assay, Co-Immunoprecipitation Assay