2 iminothiolane traut s reagent

BMP-2 was thiolated following an adapted protocol.^{30 (link)} Briefly, 2-iminothiolane (Traut’s reagent, Thermo Fisher Scientific) was reacted with recombinant, CHO-derived human/murine/rat BMP-2 (Peprotech, Catalog Number 120–02C) at a 4M excess for 1 h in buffer at room temperature to produce the product BMP2-SH. Assessment of bioactivity was performed using the SMAD 1/5/8 luciferase reporter HEK293 cell line (Signosis, SL-0051) per manufacturer provided instructions. Briefly, HEK293 cells were expanded in complete growth medium containing Dulbecco’s modified Eagle media (DMEM, Invitrogen), 10% fetal bovine serum (FBS, Atlanta Biologicals), and 1% penicillin/streptavidin (Corning) to ~90% confluency. Cells were trypsinized (Life Technologies), seeded into a 96-well plate at a concentration of 1×10⁴ cells per well, and incubated at 37^oC with 5% CO₂ overnight. The next day, cells were incubated for 16 hours in media containing DMEM, 0.1% FBS and increasing concentrations of BMP-2 or BMP2-SH. Cells were washed with PBS and lysed with lysis buffer. Lysates were treated with luciferase substrate (Promega) and luminescence was recorded on a plate reader (BMG LabTech). Luminescence was subtracted from a blank containing lysis buffer and luciferase substrate.

Heterobifunctional polyethylene glycol (NHS-PEG-PDP; 2 kDa) was purchased from JenKem Technology USA Inc. (Plano, TX). E. coli strains (TOP10 and BL21star (DE3)), pEXP-5-NT/TOPO^® TA expression kit, fetal bovine serum (FBS), PBS, Dulbecco's Modified Eagle Medium (DMEM) and Hoechst 33342 were purchased from Invitrogen (Carlsbad, CA). Traut's reagent (2-iminothiolane), Isopropyl-β-thiogalactopyranoside (IPTG) and kanamycin were from Fisher Scientific (Pittsburg, PA). T4 DNA ligase and DNA restriction enzymes (NheI and EcoRI) were purchased from New England Biolabs (Ipswich, MA), and PCR primers were from Integrated DNA Technologies Inc. (Coralville, IA). Rabbit reticulocyte lysate assay kit and luciferase assay kit were purchased from Promega Corporation (Madison, WI), and BCA assay kit was from Bio-Rad Laboratories (Hercules, CA). Melittin (from honey bee venom) and Rhodamine B isothiocyanate (TRITC) were obtained from Sigma-Aldrich (St. Louis, MO). Cell proliferation kit II (XTT) was purchased from Roche Applied Science (Indianapolis, IN).

Tetraethyl orthosilicate (TEOS), 1H-imidazole-4-carboxylic acid, thionyl chloride (SOCl₂), Traut’s reagent (2-iminothiolane), Triton X-100, acetone, ethanol, glutathione (GSH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and ammonia (30% in water) were purchased from Fisher Scientific, USA. Hexanol, cyclohexane, and (3-aminopropyl)triethoxysilane (APTES) were bought from Tokyo Chemical Industry Co., Ltd., USA. Triethylamine (TEA) and dimethyl sulfoxide (DMSO) were purchased from Alfa Aesar, USA. Bis[3-(triethoxysilyl)propyl]-disulfide (BTPD) was purchased from Gelest, Inc., USA. Methoxy-poly(ethylene glycol)-silane (mPEG-silane, M_n = 5000), amine-poly(ethylene glycol)-silane (NH₂-PEG-silane, M_n = 5000) and maleimide-poly(ethylene glycol)-silane (Mal-PEG-silane, M_n = 5000) were purchased from Biochempeg Scientific Inc., USA. All-trans-retinoic acid (ATRA) and 4-aminophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside were purchased from Santa Cruz Biotechnology, USA. Nuclear localization signal (NLS)-tagged Streptococcus pyogenes Cas9 nuclease (sNLS-SpCas9-sNLS) was obtained from Aldevron, USA. In vitro transcribed single guide RNAs (sgRNAs) and ssODNs were purchased from Integrated DNA Technologies, Inc., USA.