Anti ciita

Protein extracts were obtained by lysing cells. Samples were fractionated by SDS-PAGE and transferred to polyvinylidene-fluoride (PVDC) membrane. After blocking with 5 % milk in PBST (phosphate-buffered saline with 0.1 % tween 20) for 1 h at room temperature, the membranes were probed with the corresponding primary and secondary antibodies. And the following antibodies were used: anti-HLA DR+DP+DQ (1:200, Abcam), anti-CIITA (1:200, Santa Cruz Biotechnology) and anti-β-Actin (1:5000, HuanAn).

Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche). FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blot analyses were performed with anti-FLAG, anti-β-actin, anti-HA, anti-GFP (Sigma), anti-CIITA (Santa Cruz), anti-PRMT1, and anti-methylated arginine (Abcam) antibodies.

Western-Blotting was performed as described before^{21 (link)}. Samples (20 µg protein extract) were separated on 10% SDS-polyacryamide gels followed by semi-dry blotting onto PVDF membranes (Roche, Basel, Switzerland). Membranes were blocked for 1 hour at room temperature in tris buffered saline (TBS) (Bio-Rad, Hercules, USA) and incubated overnight at 4 °C with one of the following primary polyclonal antibodies: anti-VCAM-1, anti-ICAM-1 (both from R&D Systems, Wiesbaden, Germany) or anti-CIITA (Santa Cruz Biotechnology, Dallas, USA). Hereafter, the membranes were thoroughly washed with TBS 0,1% Tween-20 and incubated with the appropriate horseradish peroxidase conjugated secondary antibody (Jackson ImmunoResearch, Baltimore, USA). Proteins were visualized using enhanced chemo luminescence technology (Pierce, Rockford, USA). To confirm equal protein loading, membranes were stripped and re-probed with monoclonal anti-β-actin (Santa Cruz Biotechnology, Dallas, USA) antibody.