Anti ha conjugated magnetic beads

SCID.adh cells and HeLa cells, or HEK293T cells transfected with expression constructs using Lipofectamine 2000 one day earlier, were harvested using Pierce lysis buffer supplemented with protease (Thermo Scientific, 78425) and phosphatase inhibitors (EMD Millipore, 4906845001). Lysates were incubated on ice for 30 min, with vigorous vortexing every 10 min, and then pre-washed with Pierce Protein A/G agarose slurry for 1 h at 4°C to block non-specific binding. HA-tagged proteins were subjected to IP at 4°C overnight using antibody-agarose beads (Clontech, 631207) followed by three washes with Pierce lysis buffer or were collected anti-HA-conjugated magnetic beads followed by TBST washes (Thermo Scientific, 88836).

The following antibodies were used in this study: anti-Sec61α (1:5000), anti-Sec61β (1:5000), anti-TRAPα (1:5000), anti-SRα (1:5000), anti-FLAG (1:1000), and anti-PrP-A antibodies (1:5000), which have been previously described [7 (link),10 (link),18 (link)]. Anti-clanexin (1:1000), anti-BiP (1:1000), and anti-calreticulin antibodies (1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-PDI (1:5000) and anti-HA-conjugated magnetic beads were obtained from Thermo Fisher Scientific (Waltham, MA, USA). A PrP-specific 3F4 antibody (1:10,000) was purchased from BioLegend (San Diego, CA, USA). Anti-FLAG (M2)-conjugated magnetic beads, FLAG peptide, DTT, thapsigargin (Tg), and all chemicals for biochemical analyses were purchased from Sigma-Aldrich Korea (Seoul, Korea).