The HEK293 (293) cell line stably co-transfected with rat nAChR subunits α4 and β2 are well characterized [6 (link),11 (link),12 (link)], and they were maintained as described previously [9 (link),10 (link),14 (link)]. This cell line also expresses TNFR1 (only weak expression of TNFR2 is detected) and no additional human nAChR subunits or acetylcholine synthetic enzymes are detected [9 (link),10 (link)]. As before treatments were conducted 48 hours after cell plating [9 (link),10 (link),14 (link)]. The source and optimal concentrations of the inhibitors used are as follows: From Sigma; 1 μM nicotine (nicotine hydrogen tartrate); 250 μM choline (choline chloride); hemicholinium-3 (HC3; 50 μM). From Biosource; 25 ng/ml human TNFα. From Tocris Bioscience: 1,2,3,4,5,6-Hexabromocyclohexane (1-6Hex; 50 μM), tyrphostin 2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG-490; dose as in text), Cucurbitacin I (100 nM); GSK690693 (5 μM), Lestaurtinib (300 nM), Ly294002 (10 μM), PI 828 (20 nM), SB2020190 (10 μM), Wortmannin (3 μM), ZM 39923 (50 μM), ZM449829 (50 μM). From Cayman Chemical: AS-605240 (20–100 nM); PIK-75 (50 μM). Most inhibitors were dissolved in either DMSO or ethanol as the carrier and in all cases the carrier was added to cultures at the same concentration as before [9 (link),10 (link),14 (link)].
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