X SDSloading buffer and loaded onto a SDSPAGE. Afterwards, the gel was Coomassie stained and also the visible bands reduce out and alyzed by mass spectrometry.Cell culture, transfection, ATPDepletion and infectionHEKT (ATCC# CRL) cells were grown on mm or mm tissue culture dishes (Nunc) at uC and CO in DMEM with FCS containing IUml Penicillin and mgml Streptomycin (all from Gibco). Transfection was carried out at about confluence using the Calcium phosphate approach following regular procedures. Through serum starvation situations HEKT cells had been incubated in DMEM as described above without the need of FCS for hours. For serum starvation medium was exchanged hours before harvesting to serumfree DMEM with IUml Penicillin and mgml Streptomycin. For MEK inhibition hours following transfection HEKT cells were serumstarved and hours later had been treated with mM MEKinhibitor (see above beneath Chemical compounds). minutes later cells have been harvested. For ATPdepletion of HEKT cells the medium was exchanged for PBS (Gibco) containing mM deoxyglucose and mM rotenone (each from Sigma). JA. (ATCC# TIB) cells had been grown in RPMI supplemented with FCS, IUml Penicillin and mgml Streptomycin and additiol mmol Glutamine (all from Gibco). Upon infection medium of JA. cells had been changed to antibiotic cost-free RPMI. Yersinia AN3199 strains and Infection Yersinia enterocolitica strains 
utilized in this study are deltaYopM, a derivative in the Yersinia enterocolitica Serotype O: strain WA harbouring the virulence plasmid pYVO, in which the YopM gene had been replaced by a kamycin resistance cassette, precisely the same deltaYopM strain complemented together with the D construct YopMCBPSBP in pACYC (described beneath plasmids) (deltaYopM(pYopMCBPSBP)), deltaYopM complemented with YopM in pACYC (deltaYopM(pYopM)), WAC(pTTSS), the virulence plasmid cured Yersinia enterocolitica strain WAC harbouring the plasmid pTTSS encoding the TTSS secretiontranslocation apparatus of WA but no Yop PubMed ID:http://jpet.aspetjournals.org/content/135/3/275 effector gene and WAC(pTTSS) complemented with YopM in pACYC (WA A single one.orgMass spectrometryThe proteins (bands cut out from Coomassie stained SDels) had been reduced with mM 
dithiothreitol (DTT) at uC for min, the cysteine residues modified with iodacetamid ( mM, ambient temperature, min inside the dark) along with the protein ingel digested with trypsin (conditions: ng trypsinml (sequencing grade modified trypsin, Promega, Madison, USA) in mM NHHCO, uC, h). Immediately after digestion the gel pieces have been repeatedly extracted ( acetonitrile formic acid), the combined extracts dried down in a vacuum concentrator and redissolved in methanol formic acid. ml of sample was mixed with an equal volume of matrix option (saturated resolution of ON123300 cyanohydroxycinmic acid (HCCA) in water acetonitrile. trifluor acetic acid (TFA)) and applied onto a MALDI target by the drieddroplet process. Peptide mass fingerprint data had been determined on a MALDITOF mass spectrometer (REFLEX IV, Bruker Daltonics, Bremen, Germany) in reflector mode. Database searches have been done using the Mascot search algorithm version. (Matrix Sciences, London, UK) applying the following parameter: mass tolerance: ppm, one missed tryptic cleavage permitted, fixed modification: carbamidomethyl cysteine, variable modification: monooxidized methionine, database searched: NCBI nr, searches limited to mus musculus.Bacterial protein expression, purification and pulldown assayBacterial protein expression and purification had been done basically as described. Briefly, the empty pGEXKG or YopM in pGEXKG have been transformed into BL E. coli.X SDSloading buffer and loaded onto a SDSPAGE. Afterwards, the gel was Coomassie stained and also the visible bands reduce out and alyzed by mass spectrometry.Cell culture, transfection, ATPDepletion and infectionHEKT (ATCC# CRL) cells have been grown on mm or mm tissue culture dishes (Nunc) at uC and CO in DMEM with FCS containing IUml Penicillin and mgml Streptomycin (all from Gibco). Transfection was carried out at approximately confluence applying the Calcium phosphate process following common procedures. Throughout serum starvation situations HEKT cells had been incubated in DMEM as described above without having FCS for hours. For serum starvation medium was exchanged hours before harvesting to serumfree DMEM with IUml Penicillin and mgml Streptomycin. For MEK inhibition hours immediately after transfection HEKT cells were serumstarved and hours later have been treated with mM MEKinhibitor (see above beneath Chemicals). minutes later cells were harvested. For ATPdepletion of HEKT cells the medium was exchanged for PBS (Gibco) containing mM deoxyglucose and mM rotenone (each from Sigma). JA. (ATCC# TIB) cells have been grown in RPMI supplemented with FCS, IUml Penicillin and mgml Streptomycin and additiol mmol Glutamine (all from Gibco). Upon infection medium of JA. cells have been changed to antibiotic absolutely free RPMI. Yersinia strains and Infection Yersinia enterocolitica strains utilized in this study are deltaYopM, a derivative on the Yersinia enterocolitica Serotype O: strain WA harbouring the virulence plasmid pYVO, in which the YopM gene had been replaced by a kamycin resistance cassette, the same deltaYopM strain complemented with all the D construct YopMCBPSBP in pACYC (described under plasmids) (deltaYopM(pYopMCBPSBP)), deltaYopM complemented with YopM in pACYC (deltaYopM(pYopM)), WAC(pTTSS), the virulence plasmid cured Yersinia enterocolitica strain WAC harbouring the plasmid pTTSS encoding the TTSS secretiontranslocation apparatus of WA but no Yop PubMed ID:http://jpet.aspetjournals.org/content/135/3/275 effector gene and WAC(pTTSS) complemented with YopM in pACYC (WA A single one particular.orgMass spectrometryThe proteins (bands cut out from Coomassie stained SDels) have been lowered with mM dithiothreitol (DTT) at uC for min, the cysteine residues modified with iodacetamid ( mM, ambient temperature, min inside the dark) and the protein ingel digested with trypsin (conditions: ng trypsinml (sequencing grade modified trypsin, Promega, Madison, USA) in mM NHHCO, uC, h). Soon after digestion the gel pieces were repeatedly extracted ( acetonitrile formic acid), the combined extracts dried down inside a vacuum concentrator and redissolved in methanol formic acid. ml of sample was mixed with an equal volume of matrix remedy (saturated remedy of cyanohydroxycinmic acid (HCCA) in water acetonitrile. trifluor acetic acid (TFA)) and applied onto a MALDI target by the drieddroplet system. Peptide mass fingerprint data were determined on a MALDITOF mass spectrometer (REFLEX IV, Bruker Daltonics, Bremen, Germany) in reflector mode. Database searches had been completed with all the Mascot search algorithm version. (Matrix Sciences, London, UK) using the following parameter: mass tolerance: ppm, a single missed tryptic cleavage allowed, fixed modification: carbamidomethyl cysteine, variable modification: monooxidized methionine, database searched: NCBI nr, searches restricted to mus musculus.Bacterial protein expression, purification and pulldown assayBacterial protein expression and purification were carried out primarily as described. Briefly, the empty pGEXKG or YopM in pGEXKG have been transformed into BL E. coli.