Anti mlh1 g168 728

The HEK293T cell line, pcDNA3-MLH1, and pSG5-PMS2 have previously been described [26] . The Q5 Site directed mutagenesis system (New England Biolabs, Frankfurt, Germany) was used to generate missense variants with appropriate primes according to the manufacturer's protocols. Direct sequencing was used to confirm all of the plasmids that were created [14].
HEK293T cells were transfected as previously described [28] . In brief, at 50-70% confluence, HEK293T cells were transiently transfected in 10 cm round dishes with expression plasmids (1 g/ml, respectively) using 10 l/l of the cationic polymer polyethylenimine (Polysciences, Warrington, PA; stock solution 1 mg/ml). Cells were prepared for confocal laser scanning microscopy or protein extraction after 48 hours [25, 27, 28] .
The extracts were examined using SDS-PAGE and immunoblotting with anti-MLH1, G168-728 from BD Biosciences, as well as anti-PMS2, E-19, and anti-beta-Actin, C2 from Santa Cruz Biotechnologies. A Fuji LAS-4000 mini camera and Multi Gauge v3.2 were used to detect and quantify chemiluminescence signals (Immobilon, Millipore) [14] .

Anti-MLH1 (G168-728) and anti-PMS2 (A16-4) were purchased from Pharmingen (BD Biosciences, Heidelberg, Germany), anti-beta Actin (Clone AC-15) was from Sigma-Aldrich (Munich, Germany). Anti-phospho-AKT-substrate (23C8D2), hereinafter The pcDNA3.1+/MLH1 and pcDNA3.1+/PMS2 expression plasmids were described previously [12] . In addition, the MLH1-variants MLH1 S477A , MLH1 D478A and MLH1 E480A were generated by site-directed mutagenesis (for detailed primer information see supplementary table 1) and overexpressed in HEK293T cells.
All plasmids were confirmed by sequencing. All oligonucleotides were purchased from Sigma-Aldrich (Munich, Germany). pEGFP_C1 plasmid (negative control plasmid for the MMR assay) was purchased from Clontech Laboratories.