Membranes have been at first blocked, followed by e posure to cell lysate. Immediately after washing, e po sure to biotin conjugated cytokine antibody and HRP conjugated streptavidin, cytokines had been detected employing typical chemiluminescent solutions. The proce dure was carried out 3 times. Determination of MIP 2 e pression by Mesangial Cells MC had been initially seeded unto plastic dishes in DMEM supplemented with 10% FBS. Subsequently, cultures were serum starved overnight, fol lowed by incubation with L cysteine or Hcy for 24 hrs at 37 C. Cells have been harvested and total RNA was isolated by estab lished procedures. Following cDNA synthesis, qPCR was carried out using an iQ SYBR Green kit. Detection of MIP 2 Protein in Mesangial cells Cultures have been serum starved overnight, followed by incu bation with L Cys or Hcy for 24 hrs at 37 C.

Subsequently, cells have been washed with phosphate buffered saline and harvested below non denaturing disorders by incuba tion Brefeldin_A with lysis buffer. Following centrifugation, the supernatant was transferred to a fresh microcentrifuge tube as well as the protein concentration was measured with Bio Rad protein assay reagent. Protein was separated on a SDS Webpage gel. Soon after electroblotting to a nitrocellulose membrane, membranes have been incubated with 25 ml of blocking buffer after which above night at four C with rabbit polyclonal macrophage inflam matory protein two antibody in twenty ml of antibody dilution buffer with gentle rocking. Membranes were washed 3 occasions with TTBS then incubated with HRP conjugated anti rabbit secondary antibody in 20 ml of anti body dilution buffer.

Following three even more TBS washes, the membrane was incubated with ECL Chemilumines cence Reagent and then e posed to ray film. Immune comple es were removed through the membrane by deal with ment with stripping buffer. Subsequently, protein loading was assessed by re blotting with anti actin antibody and an HRP conjugated anti rabbit second ary antibody. Pro tein bands have been quantified making use of BioRad Quantity A single computer software package. To be able to study the effect of kinase inhibitors on MIP 2, MCs have been incubated during the presence of Hcy with or without inhibitors U0126, SB203580 and LY294002 for 24 h at 37 C. Subsequently, cells had been washed with PBS and har vested underneath non denaturing ailments by incubation with lysis buffer as described above. MIP 2 protein was quantified following detection by western blot as described above.

Immunofluorescence Microscopy for MIP two MCs have been at first plated onto sterile two chambered slides e actly as described for other e periments above. After incubation in the presence of Hcy with or with no kinase inhibitors, cells were washed and fi ed. Following PBS washes, cells were permeabilized, washed once more with PBS and incubated with blocking remedy for 60 minutes at room temperature. The cells were subsequently incubated with rabbit poly clonal MIP two antibody constituted in blocking option.