2 thio utp

mRNA transcripts of Tlr1, 2 and 6 were produced as previously described [17 (link)]. In brief, T7-promoter-containing pVAX.A120-vectors encoding for Tlr1, 2 and 6 were linearized and transcribed in vitro into chemically modified mRNA, incorporating 25% 2-Thio-UTP and 25% 5-Methyl-CTP (TriLink Bio Technologies) using the T7 MEGAscript kit (Ambion). Modified mRNA was purified using the MEGAclear kit (Ambion) and dissolved in RNase-free DEPC-water.

Fura‐2 AM was from Invitrogen (Paisley, UK). Fluo‐8 AM was from Stratech Scientific (Suffolk, UK). MRS2365 ((N)‐methanocarba‐2‐methylthio‐adenosine‐5′‐diphosphate), MRS2179 (2′‐deoxy‐N⁶‐methyladenosine 3′,5′‐bisphosphate), U73122 (1‐[6‐[[(17β)‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino]hexyl]‐1H‐pyrrole‐2,5‐dione), U73343 (1‐[6‐[[(17β)‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino]hexyl]‐2,5‐pyrrolidinedione), SKF96365 (1‐[2‐(4‐methoxyphenyl)‐2‐[3‐(4‐methoxyphenyl)propoxy]ethyl‐1H‐imidazole hydrochloride) and BTP‐2 (N‐[4‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]phenyl]‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide) were from Tocris (Bristol, UK). Fibronectin was from Merck Millipore (Watford, UK). Bovine serum albumin was from Europa Bioproducts Ltd (Cambridge, UK). 2′‐amino‐UTP (2′‐amino‐2′‐deoxyuridine‐5′‐triphosphate) and 2′‐thio‐UTP (2′‐thio‐2′‐deoxyuridine‐5′‐triphosphate) were from Trilink Biotechnologies (San Diego, CA, USA). Thapsigargin was from Bio Techne (Abingdon, UK). qPCR primers and related reagents were from Qiagen (Crawley, West Sussex, UK). All other reagents, including 2‐aminoethoxydiphenyl borate (2‐APB), ADP, ATP, probenecid, UDP and UTP were from Sigma‐Aldrich (Gillingham, UK). The properties of the drugs used are summarized in Table S1.