Dylight800

Protein isolation was performed by scraping the co-cultured cells into 50 μl of lysis buffer (10 mM Tris-HCl, 10 mM Hepes, 150 mM NaCl, 5 mM EDTA, Complete EDTA-free protease inhibitor cocktail (Roche), 1 μg/ml pepstatin (Sigma), 0.5% NP40 and 1% Triton) per well, pooling 12 wells. Samples were homogenized using 1.4 mm-sized zirconium oxide beads and the Precellys 24 homogenizer (Peqlab), and protein concentrations were measured using Bradford Ultra (Expedeon) and the Infinite M200 Pro plate reader (Tecan). 20 μg of proteins were loaded on the SDS-PAGE gels. We used 4–15% gradient Mini-PROTEAN_TGX precast gels (Bio-Rad) for the detection of α-Tubulin (Neomarkers, Thermo Scientific, 1:2000), collagen (F1C3, 1:1000) and Tnc (KAF14, 1:1000). Semi-dry blotting was performed at 200 mA for 1h and subsequently at 120 mA for 1 h using the Trans-Blot SD Blotter (Bio-Rad). Blocking was performed using 1x Roti-Block (Roth) and primary antibodies were incubated overnight at 4°C. After washing with TBS-T, anti-mouse DyLight-800 and anti-rabbit DyLight-680 secondary antibodies (Cell Signaling) were used at 1:15000 and after washing, the signals were measured using the Odyssey CLx infrared imaging system (LI-COR). Quantification of 3 technical replicates was performed using the Image Studio Lite Western blot analysis software. Statistics: one-way Anova with Bonferroni post-hoc test.

Following a previously established protocol (23 ), auto-mono-ADP-ribosylated PARP10cd was obtained by incubating 10 μM purified PARP10cd in 1 mM NAD+ for 20 min at 37˚C in Reaction Buffer (50 mM HEPES pH 8.0, 0.15 M NaCl, 0.2 mM TCEP, 0.02% NP-40). The de-ADP-ribosylation reaction was performed by incubating purified RuV macrodomain with mono-ADP-ribosylated PARP10cd at 37˚C in Reaction Buffer at equimolar ratios (1 μM). The reaction was terminated at 0, 1, 2, 4, 8, 16, 32 or 64 min by adding SDS-PAGE sample loading buffer and incubating at 95˚ for 5 min. Following separation SDS-PAGE on a 4-20% gradient gel, the proteins were transferred to a PVDF membrane and the de-MARylation activity determined by Western blotting. The membrane was blocked in 5% milk in PBS, 0.05% Tween-20. The membrane was incubated for 3 h at 22˚C in Anti-mono-ADP-Ribose Binding Reagent (Sigma, MABE1076, RRID:AB_2665469; 1:3,000 dilution) in place of a primary antibody. The secondary antibody was anti-rabbit immunoglobin (Cell Signaling Technology, 5151S, RRID:AB_10697505, DyLight® 800; dilution 1:10,000 dilution, 30 min at 22˚C). Blots were imaged with the near-infrared system of an Odyssey fluorescent scanner (LI-COR Biosciences) after washing with PBS and water.