Ngcontent121Page 13 ofdigested with XhoI and Asp718 and cloned into pcDNA
Ngcontent121Page 13 ofdigested with XhoI and Asp718 and cloned into pcDNA5 FRTTOspecial-WTgp130-YFP making the plasmid pcDNA5FRTTOspecial-CAgp130-YFP. For generation of mCherry-tagged receptor constructs mCherry-cDNA was amplified by PCR working with the plasmid pcDNA5FRTTOspecial-Stat3-mCherry (previously constructed in our lab) as being a template: senseP 5′-CCG GTC GCG ATA TCG GTG AGC AAG GGC GAG GAG-3′, antisenseP 5′-AGA GTC GCG GAT CCT TTA CTT GTA CAG CTC GTC C-3′. The PCR product was subcloned into pCR2.1-TOPO and the resulting plasmid was digested with EcoRV and BamHI. The produced fragment was cloned into pcDNA5FRTTOspecial-WTgp130-YFP resulting in the plasmid pcDNA5FRTTOspecialWTgp130-mCherry. For generation of mCherry-tagged CAgp130 the fragment that resulted from XhoI and Asp718 digestion of pCR2.1-Topo-CAgp130 (see above) was cloned into pcDNA5FRTTOspecial-WTgp130mCherry producing the plasmid pcDNA5FRTTOspecialCAgp130-mCherry. For generation of add-back mutants of CAgp130 previously constructed plasmids were made use of [13]. New constructs were created by three-fragment-ligation. The backbone was created by XhoI and EcoRV digestion of pcDNA5FRTTOspecial-WTgp130-YFP. The extracellular part of CAgp130 was isolated upon XhoI and EcoRI digestion of pcDNA5FRTTOspecial-CAgp130YFP. The intracellular a part of gp130 harboring mutated Tyr-residues was generated by EcoRI and EcoRV digestion on the preexisting constructs. Following constructs were produced: pcDNA5FRTTOspecial-CAgp130-6FYFP, -CAgp130-Y915-YFP, -CAgp130-Y905-YFP, -CAgp130Y814-YFP, -CAgp130-Y767-YFP, -CAgp130-Y759-YFP and -CAgp130-Y683-YFP. For generation of your K44A dynamin construct the plasmid pMSCV-IRES-GFP (kindly supplied by Dr. N. Chatain) was digested with EcoRI and SalI as well as generated fragment was cloned into EcoRI and XhoI digested pcDNA3.1(). SalI and XhoI produce complementary overhangs and upon ligation each restriction websites are destroyed resulting in the plasmid Plasmid pcDNA3.1()-IRES-GFP was digested with BamHI and EcoRI delivering the backbone for the subsequent cloning stage. The construct (kindly offered by Dr. S. W ler) was digested with BamHI and NheI to isolate the N-terminal part of HA-hu-dynamin-K44A. To generate an EcoRI internet site and amplify the C-terminal a part of HA-hu-dynamin-K44A PCR was performed on pcDNA3.1(-)-HA-hu-dynaminK44A: senseP ETB Storage & Stability 5′-CGA GCA AGC ATA TCT TTG CC3′, antisenseP 5′-GCA TCG AAT TCT TAG AGG TCG AAG GGG GGC-3′. The plasmid was created by three-fragmentligation. All constructs have been verified by sequencing.Cell culture, iNOS Molecular Weight transient and stable transfectionHEK293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax (Gibco, Germany) supplemented with 10 FCS (Lonza, Germany), 60 mgl penicillin and 100 mgl streptomycin (Gibco, Germany). For HEK293 cells stably expressing IL-6R (kindly supplied by Dr. Anna Dittrich) medium was supplemented with 2 mgl Puromycin (Invivogen, CA, USA). Transient transfections were performed with TransIT-LT-1 transfection reagent (Mirus, Madison, USA). T-REx-293 cells had been stably tranfected using the Flp-In system (Invitrogen). Antibiotics for generation and upkeep of stable cell lines blasticidin, zeocin, hygromycin B were purchased from Invivogen.Planning of cell lysates, SDS-PAGE and immunoblottingReceptor expression was induced with 20 ngml or 0.five gml dox. Stimulation was carried out with 200 Uml IL-6 and 0.