Pegfp c2

By C-17 protocol, 5% SRGF and 10% FBS ASC (5 × 10⁵ cells) were nucleofected to deliver with 1 μg of a mammalian expression vector pEGFP C2 (AddGene, Watertown, MA, USA) containing the CD63 gene sequence. To evaluate CD63 NF efficiency, the percent fraction of GFP positive cells was evaluated by flow cytometry: thus, as a control for such experiments, NF of ASC without DNA was performed. Overexpression of CD63 was also evaluated by Western blot analysis. In such experiments, NF of 1 μg of the mammalian expression vector pEGFP C2 (AddGene) containing the CD63 gene sequence was performed. As control for Western blot analysis, NF of the empty pEGFP C2 vector was performed. Then, 24 h after transfection, cells were collected and lysed by NP40 Cell Lysis Buffer (Thermo Fisher Scientific). After separation by Mini Protean TGX Precast Gel (gradient 4–20%) proteins were blotted on nitrocellulose membranes. Expression of CD63 was analyzed by Western blot using anti-human CD63 (Invitrogen, Carlsbad, CA, USA) (dilution 1:1000) and, as loading control, anti-human GAPDH (dilution 1:1000) (Calbiochem, San Diego, CA, USA) antibodies. Intensity of CD63 and GAPDH Western blot bands were quantified by ImageJ software (National Institutes of Health). For comparison between electroporated 10% FBS and 5% SRGF ASC, CD63 expression level was defined as ratio between CD63 and GAPDH band intensity.

For transfection assays, the CERKL cDNA transcript variant 1 (NM_201548.4) was cloned into pEGFP-C2 (Addgene) (CERKL-GFP) and into modified versions of pcDNA (Clontech Laboratories, Inc.), which add a C-terminal hemagglutinin (CERKL-HA) or a Flag (CERKL-Flag) tag. The mutant encoding amino acids 1 to 256 tagged with HA was cloned into pcDNA (CERKL 1–256). All constructs were generated using standard recombinant DNA techniques. A plasmid expressing Flag-GFP was kindly provided by Dr. A.T. Ting (Mount Sinai School of Medicine, NY, USA). A CERKL-C125W point mutant was generated using the QuikChange XL site-directed mutagenesis kit (Stratagene/Agilent Technologies), according to the manufacturer's instructions. To produce recombinant MBP-CERKL fusion proteins in E. coli, the cDNA encoding the full length CERKL protein or its amino acids 1–256 and 252–532 were inserted into the pDEST-His-MBP vector (Addgene) using the Gateway recombination cloning kit (Invitrogen Life Technologies). All constructs were confirmed by sequence analysis.

All plasmids were transfected with Lipofectamine 2000 (Life Technologies-Invitrogen, USA) according to the manufacturer’s instructions. H1.2, H1.3 and H1.4 cDNAs were amplified and cloned into the p3× FLAG-CMV-10 vector (Addgene, USA). Full-length H1.2 and various fragments (N-terminal domain, 1–35 aa; globular domain, 36–112 aa; C-terminal domain, 113–213 aa; ΔC1, 1–179 aa; ΔC2, 1–112+180–213 aa; ΔC, 1–112 aa) were cloned into the pEGFP-C2, pGEX-4T3 or pET28a vectors (Addgene, USA). The full-length FLAG-ATM expression construct was purchased from Addgene, USA. GST-ATM fragments (F1, 1–247 aa; F2, 250–522 aa; F3, 523–769 aa; F4, 722–1102 aa; F5, 1098–1371 aa; F6, 1245–1435 aa; F7, 1239–1770 aa; F8, 1764–2138 aa; F9, 2141–2428 aa; F10, 2427–2841 aa; F11, 2842–3056 aa; F12, 2682–3012 aa) were provided by Dr. Fabrizio d’Adda di Fagagna (FIRC Institute of Molecular Oncology Foundation, Italy). Site-specific mutations of H1.2 (T126/146/165A, T126/146/165E, E115A, S173A, S188A) were generated using a site-directed mutagenesis kit (Vazyme, China).