Cell lines, Islets and Embryonic Tissue bTC3 (bTC), aTC1.3 (aTC), L and HEK293F (HEK293) cells were cultured in DMEM with 10% FBS and 1% pen/strep. bTC, L and HEK293 cells were cultured with 5 g/L glucose, and aTC with 1g/L glucose. Islets from wild type NMRI mice were isolated using collagenase digestion and were grown for 5 days in RPMI 1640 with 10%FBS and 1% pen/strep. All embryonic mouse tissue were from wild type NMRI mice, expect for the Neurog3- knockout [53]. For analysis of embryonic pancreas tissue time- mated mice were sacrificed at the indicated gestational stage, the embryos were removed and the embryonic pancreas dissected under microscope using a small knife and forceps. Cloning and Plasmids Wildtype mouse Pdx1 (Pdx1WT) cDNA were obtained as an imageclone from Open Biosystems (GenBank: BC103581.1). This cDNA was amplified by PCR and inserted in the pENTR/D vector (Invitrogen) and then transferred to our previously published expression vector pCGIG5 [44], for expression in eukaryotes and to the pEXP2 plasmid (Invitrogen) for expression in bacteria. To generate, the 3xFlag version we used a 3’ primer comprising the tag. To introduce point mutations we performed chimeric PCR using primers covering 12 nucleotides up and down stream of the codon being modified. Correct clones were identified by diagnostic RE digests and sequence verified (MWG, Ebersberg, Germany). As control we used the pCGIG5 vector either without insert (designated empty vector) or with EGFP. Wildtype GSK3b were cloned from Xenopus and a kind gift from Chris Wright. Cell/tissue Harvest For direct NIA and western analyzes all cell lines, tissue and islets were lysed in Tissue Extraction Reagent I (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 2 mM, Na3VO4, 1 mM NaF, 20 mM Na4P2O7, 0.02%NaN3, and proprietary detergent), from Invitrogen. Prior to use this buffer was supplemented with protease inhibitors (Complete protease inhibitor cocktail tablets, Roche). Before harvest, the cells were washed once in PBS and thoroughly aspirated. One 6-well of cells was lysed in app. 100 ml, one litter of E15.5 pancreata was lysed in 20 ml and 200 islets in 8 ml. For the Neurog3-KO, individual pancreata were isolated and snap-frozen in dry ice and kept at 280uC, while genotyping was performed. Pancreata with the three different genotypes was pooled and lysed in 5 ml buffer per four pancreata. HEK293 lysates for immunoprecipitation followed by NIA, western or mass spectrometry analysis were harvested as nuclear extracts; One 15 cm dish were washed once in PBS followed by cytolysis for 10 min. in 1.5 ml buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA and 0.4% Triton X-100, Complete protease inhibitor cocktail tablets). Then, scraped and dissociated by pippeting and centrifuged at 10.000xg for 5 min. The pellet was resuspended in 300 ml buffer B (20 mM HEPES pH 7.9, 400 mM NaCl, 2 mM EDTA and 20% glycerol, Complete protease inhibitor cocktail tablets), sheared using a 21 G needle and shaken for 1 hour at 4uC. To generate the bacterially expressed protein, 30 ml of BL21 overnight culture was added to 250 ml fresh media and grown for 3 hours before induction with 1 mM IPTG for additionally 6 hours. The resulting bacteria pellet was washed and resuspended in 12 ml TBST supplemented with protease inhibitors, followed by sonication on ice. Additionally, all lysates were kept on ice for at least 10 min, with 2 rounds of vortexing, followed by centrifugation at 16.0006g for 20 min. and then transferred to clean tubes and kept at 280uC. Post-Translational Modification of Pdx1 Immunoprecipitation (IP) and Western Blotting (WB) For IP of wildtype Pdx1, BL21 lysates and nuclear extracts from HEK293 cells transfected with empty vector or Pdx1WT were incubated with 5 mg mouse-a-Pdx1 antibody and 50 ml magnetic protein G dynabeads (Invitrogen) per ml of lysate, for 3 hours at 4uC with end-over turning. Following four washes in TBST the beads were eluted in 8 M urea supplemented with 20 mM DTT at 30uC for 60 min with shaking. For IP of 3xFlag tagged Pdx1, BL21 lysates and nuclear extracts from HEK293 cells transfected with empty vector or Pdx13xFLAG were incubated with 25 ml magnetic a-Flag beads (Sigma) per ml of lysate for 3 hours at 4uC with end-over turning. Following four washes in TBST the 25 ml beads were eluted with 125 ml of 100 mg/ml 3xFlag peptide in TBS at 37uC for 60 min with shaking. Lastly, all eluates were concentrated app. 20 fold using spin columns with a 10 K cut- off (Amicon). Pdx1 protein for mass spectrometry was prepared from nuclear extracts of HEK293 cells transfected with Pdx1WT and immuno- precipitated as described above, however elution from the beads were done by heating at 80uC with LDS sample buffer (Invitrogen) containing 20 mM DTT. Following SDS-PAGE, the gel was stained with Coomassie (Imperial stain, Pierce); the band of interest cut out, destained with ammonium sulphate and sliced into small pieces; then reduced with DTT and alkylated using chloroacetamide. Finally, the proteins were digested overnight with trypsin, loaded onto StageTips and subjected to mass spectrometric analysis. Western blots were done using precast NuPAGE Novex Bis-Tris gels and blotted onto PVDF membranes (Invitrogen). All membranes were stained with Ponceau S to verify uniform blotting. Destaining was done with 5% acetic acid. Blocking and antibody diluent were 5% skim milk with 0.1% tween-20. The antibodies were applied for 1 hour at room temp or overnight at 4uC. Detection was done with Supersignal west pico from Pierce. Afterwards, when appropriate the membranes were incubated overnight in skim milk with 0.02% sodium azide and then reproped with another primary antibody of a different species. Cell line Transfections For transfection of aTC and L cells, plasmid DNA was purified from 40 ml overnight TOP10 bacteria culture using the S.N.A.P. kit from invitrogen. All pointmutants were transfected into each cell line in at least two different cell passages. This was done in 6- or 12-well plates using Lipofectamine2000 according to manufac- tures instructions. For transfection of HEK293 cells, plasmid DNA was isolated from 600 ml overnight culture using the Megaprep kit, from Invitrogen. HEK293 cells were transfected with empty vector or Pdx1WT in 15 cm dishes using Lipofectamine2000 according to manufactures instructions. Antibodies Primary goat-a-Pdx1 was a kind gift from Chris Wright and used at 1:50.000 for western, 1:10.000 for immunofluorescence and between 1:50 and 1:500 for NIA. Primary mouse- a-Pdx1 from the Hybridoma bank (1 mg/ml) was used at 1:10.000 for western, 1:5–1:50 for NIA and for IP at 5 mg/ml lysate. Rabbit- a-GFP from Invitrogen were used for NIA at 1:50 and mouse-a- Hsp70 from Abcam was used for NIA at 1:1.000. For western, mouse-a-b-Actin from Sigma was used at 1:25.000, guine a pig- a-insulin from Abcam at 1:100 and mouse-a-GSK3a/b from R&D at 1:500. For immunofluorescence; rabbit-a-GFP from Clonetech was used at 1:500, mouse-a-Nkx6.1 from the Hybridoma bank at 1:1000 and goat-a-FoxA2 from Santa Cruz at 1:250. All secondary antibodies were from Jackson Immunor- PLoS ONE | www.plosone.org 13 April 2012 | Volume 7 | Issue 4 | e35233