Apc coupled anti mouse antibodies

HeLa (ATCC CCL-2), CMT64.5^{41 (link)} and HEK293T (ATCC CRL-1573) cells were grown in DMEM supplemented with 10% FCS, penicillin and streptomycin. When indicated, cells were transfected using Superfect (Qiagen) following the manufacturer’s protocol. In this study the following antibodies were applied: rabbit anti-TagGFP (Evrogen); the mAb 148.3 for TAP1 detection (kindly supplied by E. Wiertz, Utrecht)^{42 (link)}; rabbit anti-calnexin (Stressgen) and mouse anti-β-actin (Sigma-Aldrich); the mAb W6/32 for the detection of fully conformed, peptide-loaded HLA-A,B,C molecules^{43 (link)}; APC-coupled anti-mouse antibodies (BD Pharmingen); anti-mouse anti-HA antibodies for flow cytometry and Western blot analysis (Sigma-Aldrich); rabbit anti-HA for immunoprecipitation (Sigma-Aldrich); anti-c-Myc and anti-FLAG clone M2 (Sigma-Aldrich); the mAb 20-8-4S specific for H-2K^b and H-2D^{b 
44 (link)}. The polyclonal anti-US6 serum was described before^{3 (link)}.

The following antibodies were applied: W6/32 (anti-pan-HLA-A,B,C assembled with β₂m and peptide, [58 (link)]), BB7.2 (anti-HLA-A2 [74 (link)]), BB7.1 (anti-HLA-B7 [74 (link)]), TT4-A20 (anti-HLA-B44 [75 (link)]), HC10 and HCA2 recognizing free HLA-B/C and HLA-A heavy chains, respectively [76 (link)], anti-CD71 (immunotech), anti-CD85j (LIR1; Miltenyi) mouse and rabbit anti-HA antibodies (Sigma), anti-ERp57 (Millipore), APC-coupled anti-mouse antibodies (BD Pharmingen). Polyclonal anti-tapasin and anti-US11 anti-sera were raised by immunization of rabbits (Genscript) with synthetic peptides (aa 418–428 and 90–103, respectively).