Cl 4b beads

Cells grown for 72 h to 100% confluence in 10 cm dishes were washed twice with ice-cold PBS prior to lysis on ice with 1 ml IP lysis buffer (125 mM NaCl, 20 mM Hepes pH 7.4, 1% Nonidet P40 substitute, 5 µg/ml pepstatin, 10 µg/ml chymostatin, 3 µg/ml leupeptin, 10 µg/ml antipain, 0.5 mM benzamadin, 0.2 mM PMSF, 0.1 kU/ml aprotinin, 1 mM Na₃VO₄, 1 mM NaF). Insoluble debris was removed by centrifugation for 15 min at 15,000 g, 4 °C. Lysates were precleared with CL-4B beads (GE Healthcare, Piscataway, NJ) for 30 min at 4 °C. As total lysate control, 5% of the cleared lysate was used. The remaining supernatant was incubated with 1-2 µg of antibody for 1 h at 4 °C. Sepharose-Protein G was added and the lysates were incubated overnight at 4 °C while rotating. The beads were washed five times with lysis buffer by centrifugation. Proteins were eluted from the beads by heating for 5 minutes in sample buffer with 100 mM DTT at 95 °C and the samples were, together with the lysates (5% of total), analyzed by SDS-PAA gel electrophoresis and Western blotting

Cells grown to confluence in 10-cm culture dishes were washed twice with ice-cold PBS and subsequently scraped on ice with 1 mL IP lysis buffer (125 mM NaCl, 20 mM Hepes pH 7.4, 1% Nonidet P40 substitute, supplemented with protease and phosphatase inhibitors as above). Cellular debris was removed by centrifugation (15 min, 15 × 10³g, 4 °C) and pre-cleared with CL-4B beads (GE Healthcare, Piscataway, NJ, USA) for 30 min at 4 °C. 5% of the cleared lysate was taken as a total lysate control. The remaining lysates were incubated sequentially with 1 µg of antibody (1 h, 4 °C) and Sepharose-Protein G (16 h, 4 °C while rotating). The beads were washed five times with IP lysis buffer by centrifugation. Proteins were eluted from the beads by boiling in Laemmli buffer (5 min, 95 °C) and analyzed by Western blotting. To compare co-immunoprecipitation of the target proteins with p120-1 and p120-3, two-tailed Student’s t-tests were performed. The variances were determined not to be significantly different using an F-test.

The hybridoma cell line B7/21 was expanded in Corning hybridigo SF medium to produce anti-HLA-DP antibodies (Abs). Protein A Sepharose beads (GE Healthcare) were used to purify the Abs and generate an immunoaffinity column (B7/21–Protein A Sepharose 2.5 mg/ml) (38 (link)). The cell pellets of the different HLA-DP-transduced K562 cell lines were lysed in 50 mM Tris–HCl (pH 8), 150 mM NaCl, 5 mM EDTA, and 0.5% ZWITTERGENT 3–12 (Merck) and supplemented with cOmplete Protease Inhibitor (Merck). The supernatant was precleared with CL4B beads (GE Healthcare) and applied to the immunoaffinity column with a flow rate of 2.5 ml/min. The bound peptide-HLA-DP complexes were eluted with 10% acetic acid (Merck) followed by the separation of peptides from the HLA-DP molecules using a 10-kDa membrane (Merck) (37 (link)).