Qproteome nuclear protein kit

ON‐TARGETplus Human HPSE siRNA‐smartpool (set of 4), ON‐TARGETplus non‐target pool siRNA, DharmaFECT 1 transfection reagent 1 were all purchased from Dharmacon (Little Chalfont, United Kingdom). Silencer Select HPSE siRNA (single) was from Thermo Scientific (Massachusetts). A Click‐iT^® Plus TUNEL assay kit and CellEvent^TM Green Detection Reagent Caspase 3/7 staining kit were from Life Technologies (California). iScript cDNA synthesis kit and SsoFast EvaGreen Supermix were purchased from BioRad (California). RNeasy Plus Mini Kit and Qproteome Nuclear Protein Kit were from QIAGEN (Hilden, Germany). High‐Capacity cDNA Reverse Transcription Kit was obtained from Applied Biosystems (California). Heparanase antibody #1453 was a gift from Israel Vlodavsky (Rappaport Faculty of Medicine, Haifa, Israel). Antibodies against Sp1 (1C6) and Histone 1 (G1) were from Santa Cruze. Anti‐caspase 3 (#9662), cleaved caspase 3 (#9661), PARP 1 (#9542) were from Cell Signalling Technology. Anti‐EGR1 (AF2818), CYR61 (MAB4055), TNFRSF12 (MAB1199) were from R&D Systems (Abingdon, UK), and anti‐GAPDH (AM4300) from Ambion. SuperSignal Duro Substrate from Thermo Scientific (Massachusetts).

Lysine-specific demethylase 1 (LSD1) enzyme activity was evaluated using the Abcam KDM1/LSD1 Activity Quantification kit (ab113459, Abcam) according to the manufacturer’s protocol. Briefly, LSD1 protein was extracted from both the nuclei of S-L TAM pairs (8 subjects) and in-vitro M1 and M2 polarized monocytes. Nuclei were isolated using a Qproteome Nuclear Protein Kit (Qiagen) following the manufacturer’s protocol. Soluble protein extracts were then subjected to the KDM1/LSD1 activity kit.

After 48 hours of drug treatment, the cultures were washed with 1× PBS, followed by the addition of the Qproteome Nuclear Protein kit (QIAGEN, San Francisco, CA) to separate nuclear and cytosolic NF-κB p65 protein. The cells were dissolved in lysis buffer (0.125 M Tris-HCl, pH 6.8, 4 percent SDS, 20% glycerol, and 2% 2-mercaptoethanol) for the western blot.

Identification of CAB063 binding partners was performed by co-immunoprecipitation. Both experimentally infected and pCI-CAB063 transfected HeLa cells were lyzed and fractionated (whole cell lysate and nuclear fraction) according to manufacturer instructions using Proteo Extract^® Subcellular Proteome Extraction Kit (Merck KGaA, Darmstadt, Germany) and Qproteome Nuclear Protein Kit (Qiagen GmbH, Hilden, Germany) as described previously (Forsbach-Birk et al., 2013 (link)). An anti PARP antibody included in the kit was used to control fractionation and identify the nuclear fraction that was used for further experiments as such (Supplementary Figure S1). 60 μg of lysate were incubated with anti CAB063 overnight, while this and further incubation steps were conducted at 4°C to reduce unspecific binding. 20 μl of sepharose beads were added thereafter and incubated for 2 h. After centrifugation and washing to remove unbound components, the sepharose-protein pellet was solubilized in Lämmli (for 1D-SDS gels) or rehydration buffer (for 2D gel electrophoresis) and heated to remove the beads prior to electrophoresis. To maximize the yield of candidate proteins, selected nuclear fractions were enriched with 2 μg of recombinant CAB063 to literally fish for eukaryotic targets.

For western blotting, the cytoplasmic and nuclear protein fractions of fibroblasts were prepared using the Qproteome nuclear protein kit (#37582, Qiagen^®, Hilden, Germany). The beta-Actin antibody was from Santa Cruz Biotechnology, Dallas, Texas, USA (#SC 1616-R) and the EGFP antibody was from Abcam (Cambridge, UK; # ab290). β-Actin and EGFP were detected using 1:1000 and 1:3000 dilution, respectively. The secondary HRP-conjugated anti-rabbit antibody (R&D Systems, Wiesbaden, Germany, #HAF008) was used at a 1:1000 dilution. Signals were visualized using the chemoluminescence ECL Western Blotting Analysis System (GE Healthcare, Chicago, IL, USA, RPN2108) and the Intas ChemoCam Western Blot Imaging System equipped with the Chemo Star Professional Software (INTAS Science Imaging Instruments, Göttingen, Germany).