Compartment protein extraction kit

Nuclear and cytosolic fractions were isolated from THP-1 and U937 cells using the Compartment Protein Extraction Kit (Millipore, Cat#2145, Billerica, MA). Nuclear and cytosolic protein concentrations were measured using the Bradford assay. The proteins were blotted with an antibody specific for RUNX2 and Lamin B1 purchase from Abcam (Cambridge, MA) at 1:1000 dilution.

ECM production was extracted using compartment protein extraction kit (Millipore). Proteomics analysis was performed using mass spectrometry, and the data were further processed using myProMS v3.6 (work in progress; https://github.com/bioinfo-pf-curie/myproms). Protein functions and ontology were evaluated using Metascape-A Gene Annotation & Analysis Resource website https://metascape.org/gp/index.html#/main/step1.

Approximately 5 × 10⁶ K562-AsCas12a-xNLS cells were collected for nuclear extraction. After an initial PBS wash, nuclear protein fractions were isolated with the Compartment Protein Extraction Kit (Millipore, Catalog #2145) according to the manufacturer’s instructions. To prepare whole cell lysates, 2 × 10⁶ cells were resuspended in Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol, and then denatured at 95 °C for 10 min. The protein extracts were separated on a 4–15% TGX gel (Bio-Rad), transferred to a nitrocellulose membrane, and analyzed by immunoblotting. All primary antibodies used at 1:1000 dilutions: FLAG (Sigma-Aldrich Cat# F1804, RRID:AB_262044), Lamin-B1 (Abcam Cat# ab16048, RRID:AB_10107828), and α-Tubulin (Sigma-Aldrich Cat# T6199, RRID:AB_477583). Secondary antibodies used: anti-Rabbit (LI-COR Biosciences Cat# 925-32211, RRID:AB_2651127), anti-Mouse (Thermo Fisher Scientific Cat# A-21058, RRID:AB_2535724). Blots were imaged on a Licor Odyssey. Relative abundance of protein was quantified via gel densitometry with ImageJ software. AsCas12a protein levels in the nuclear fraction were first normalized to Lamin-B1 levels and then to the corresponding 1x NLS samples.

Biobanked human myocardial samples were used to determine levels of ADAMTS4 expression and activity. Samples were taken from the free wall of the left ventricle of explanted hearts from patients with dilated non-ischaemic cardiomyopathy and from donor hearts that had not been used for transplantation due to no Scandinavian recipients being available. Tissue lysates were first solubilized using the compartment protein extraction kit (2145, Millipore, MA) according to the manufacturer’s instructions. ECM fractions were extracted as previously described²⁶ and reported in Supplementary Methods. The use of the samples was approved by the regional ethics committee (REK 2014/569 and 07482a).

Protein extraction was performed from dECM on day 21 using a Compartment Protein Extraction Kit (MERCKMillipore, USA). The protein pellets' solubilization and digestion were performed as previously described^{43 (link)}. In brief, dECM pellets were solubilized in a solution containing 8 M urea, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol. Cysteines were alkylated by adding iodoacetamide, and samples were deglycosylated with PNGaseF (New England BioLabs, USA, 1:100 units for 1 mg sample) and subsequently digested with trypsin/LysC (Promega, USA), at a ratio of 1:10,000 enzyme: substrate. Final digestions were done using trypsin (Worthington Biochemical Corporation, USA) at a ratio of 1:1000 (enzyme: substrate), followed by a second aliquot of trypsine/LysC (Promega, USA), at a ratio of 1:10,000 (enzyme:substrate).

Protein lysates from mouse and human LVs were extracted as previously described^{25 (link),45 (link)}, using a PBS‐based lysis buffer containing 1% Triton X‐100. Protein lysates from cell cultures were extracted using the buffer above, or a buffer containing 1% SDS for retrieval of ECM proteins. For studying LTBP1 in cytosolic and ECM protein fractions, cells were lysed using a NP-40-based lysis buffer and fractioned through centrifugation. For studying nuclear translocation of pSMAD, cells were lysed and proteins fractioned using the compartment protein extraction kit (Merck Millipore) according to the manufacturer’s protocol. Secreted proteins were harvested from the cell culture medium. Supernatants and lysates were stored at − 20 °C. N-linked oligosaccharides were enzymatically removed from glycosylated proteins using PNGaseF. Western blotting was performed using the Trans-Blot Turbo blotting system (Bio-Rad). Membranes were blocked in 5% non-fat dry milk, casein or BSA, before incubation with primary antibodies (see Supplementary Table S2) and species-specific horseradish peroxidase secondary antibodies. For full method description, see Supplementary Methods 3.9.