Additionally, MDP-induced autophagy was enhanced when PTPN22 was missing. This influence of PTPN22 on IBD associated inhibitor 17-AAG intracellular pathways could provide an explanation of how PTPN22 variants might functionally be linked with IBD. Materials and Methods Reagents and Antibodies All reagents were of analytical grade and purchased commercially. Monoclonal goat anti-PTPN22 antibody was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA) and mouse anti-��-actin antibody from Millipore (Billerica, MA).
Mouse anti-phospho-p38 (Thr180/Tyr182), rabbit anti-p38, rabbit anti-phospho-extracellular signal-regulated kinase (ERK)1/2 (Tyr42/Tyr44), rabbit anti-ERK1/2, mouse anti-phospho c-Jun N-terminal kinase (JNK; Thr183/Tyr185), rabbit anti-JNK, rabbit anti-phospho-NF-��B-p65 (Ser536), rabbit anti-NF-��B-p65 (ser276), rabbit anti-phospho-NF-��B p105 (Ser933), rabbit anti-NF-��B p105/p50, rabbit anti-phospho-NF-��B p100/p52 (Ser866/Ser870), rabbit anti-NF-��B p100/p52, and mouse anti-microtubule-associated proteins 1A/1B light chain 3B (LC3B) were obtained from Cell Signaling Technologies (Danvers, MA). Cell Culture and Vector Transduction THP-1 monocytes were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS at a density between 0.2 and 1��106 cells/ml. For experiments, cells were seed in 24 well plates at a density of 0.5��106 cells/ml 24 h before treatment. To generate THP-1 cells stably expressing either non-targeting control or PTPN22 specific shRNA, the following vectors were obtained from Sigma: pLKO.1 (control vector) and pLKO.
1-shPTPN22 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_008979″,”term_id”:”530537261″,”term_text”:”NM_008979″NM_008979.1-1233s1c1, PTPN22 targeting vecor), pMD2.G (packaging plasmid) and pCMV (envelop plasmid). Transductions were performed as described elsewhere [17] and cells cultured in RPMI 1640 supplemented with 10% FCS and 20 ng/ml puromycin (Invivogen, San Diego, CA). Stability of the knockdown was determined prior to each experiment by real-time PCR and knockdown efficiency found between 20�C40%. In addition knockdown was verified after each experiment by Western blot or real-time PCR. For NOD2 activation, MDP (Invivogen) was dissolved in DMSO and mixed with FuGene (Promega, Fitchburg, WI) in culture media for 15 min before applying on the cells. For control transfections, DMSO mixed with FuGene was used.
Generation of Bone Marrow Dendritic Cells (BMDC) Animal experiments were carried out according to Swiss animal welfare laws and were approved by the veterinary authorities of Zurich, Switzerland (Kanton Z��rich Gesundheitsdirektion Veterin?ramt, approval no. 54/2011). Due to the approval of the veterinary authorities of Z��rich, no further approval by an Institutional Animal Care and Use Committee (IACUC) or ethics committee was necessary. PTPN22 knockout mice were Batimastat kindly provided by Andrew C.
Related posts:
- The five HT induced arteriolar dilatation was enhanced These eff
- Fluorouracil Antimetabolites inhibitor avoid FCS induced crossreaction of serum insulin with the ELISA
- In present study, we investigated the relationship among integrin
- Total length cDNA encoding human WIPI was amplified by PCR from t
- Yet, the function of autophagy in cancer is still controversial