Dermal growth factor (Pepro Tech), and 0.01 g/ml of human basic

Dermal growth factor (Pepro Tech), and 0.01 g/ml of human basic fibroblast growth factor (Pepro Tech). Cells were cultured at 37 in a humidified atmosphere containing 5 CO2. All cell lines were used at low passage number not exceeding 30 passages.Western blot analysis and antibodies/lectinspLKO.1-puro vector encoding COSMC and non-target (scrambled, SCR) shRNA were purchased from SigmaAldrich (Germany). Generation of pseudotyped lentiviruses and transduction were performed as previously described [62]. Cells transduced with COSMC shRNA were selected by addition of puromycin (Sigma-Aldrich) to culture medium with a final concentration of 2 mM for at least one week.Immunoprecipitation and membrane protein extractionTotal protein concentrations were measured using BCA Protein Assay (Thermo Fischer). Samples containing 30 g total amount of protein were boiled for 5 min in Laemmli buffer and separated by SDS PAGE under reducing conditions in 4-15 Mini-PROTEAN TGX gels (BIO-RAD). Proteins were blotted on nitrocellulose membrane (Thermo Fischer). The membrane was blocked with 1x Carbo-Free Blocking Solution (Biozol) in TBS-T and subsequently incubated with serum (1:20) in TBS-T overnight. Tn antigen antibody (MA1-80055, clone BRIC111, Thermo Fischer), sTn antigen antibody (ABIN356328, clone STN218, antibodies online) and COSMC antibody (19254-1-AP, Proteintech) were diluted 1:250 in TBS-T. HSC70 (sc-7298), GAPDH (sc-32233,) Nucleolin (sc-8031), GRP78 (sc-1051), Alpha Enolase (sc-7455) and Annexin II (sc-9061) antibodies were purchased from Santa Cruz and diluted 1:1000. Her2 antibody (#2165, Cell Signaling) was diluted 1:100. Vicia villosa lectin B4 (B-1235) (VVL) and Wisteria floribunda lectin (B-1355) (WFL) were purchased from Vector Laboratories and diluted 1:125. Washing was performed 5 times with TBS-T. The membrane was incubated with a specific HRP-antibody (1:1250; Life Technologies) for 1 h at RT. After 5 times washing withTotal cell lysates were Capecitabine prepared with RIPA buffer. Lysates (0,5 mg) were pre-cleared with 40 l of protein A/ G-agarose beads (Santa Cruz) for 1 h. Protein A/G-agarose beads were added to the afore mentioned antibodies and rotated at 4 1 h. Pre-cleared proteins and beadconjugated antibodies were mixed, rotated at 4 overnight, pelleted and washed five times with RIPA buffer. An equal volume of 6xLaemmli buffer was added, and samples were boiled and separated by SDS olyacrylamide gel electrophoresis. For membrane protein extraction, harvested cells were resuspended in PBS and homogenized using a dounce homogenizer. Centrifugation at 100 g, 5 min, 4 removed undisrupted cells a second centrifugation at 100 g, 10 min, 4 extracted nuclei. After centrifugation at 20000 g for 90 min, at 4 , membranes were pelleted and the supernatant contained the post membrane fraction.Real-time PCRTotal RNA was isolated using the RNeasy Mini kit (Qiagen), reverse transcribed with Transcriptor reverse transcriptase (Roche) using an oligo-(dT) primer according to the manufacturer's protocol. The cDNA was subjected to real-time PCR amplification using primer sets for COSMC, GalNAc-transferases, T-synthase and GAPDH (Additional file 2: Table S1). Real-time PCR was prepared with Maxima SYBR Green/ROX qPCR Master Mix, 0.3 M forward and reverse primer and 750 ng cDNA and used on the Lightcycler 480 (Roche). The conditions were as follows: 95 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7500280 for 10 min, 45 cycles at 95 for 15 s, and 60 for 1 min. CT values were determined usin.