Anti dykddddk tag antibody beads

We prepared wild-type and domain-deleted hMuSKect-myc, as well as hLRP4-FLAG and FLAG-CTD for in vitro plate-binding assays and the ATF2-luciferase reporter assay, as described previously38 (link). Wild-type or mutant phMUSKect-myc, or phLRP4N-FLAG or pFLAG-CTD, was transfected into HEK293 cells in a 10-cm dish using the calcium phosphate method44 (link). We purified hMuSKect-myc with the c-myc-Tagged Protein Mild Purification Kit version 2 (MBL). We also purified hLRP4N-FLAG and FLAG-CTD with the Anti-DYKDDDDK-tag Antibody Beads (Wako). We confirmed the presence of isolated hMuSKect-myc by Western blotting with anti-myc antibody (9E10, Abcam), and hLRP4N-FLAG and FLAG-CTD with anti-FLAG antibody (M2, Sigma-Aldrich). We also confirmed the purity of isolated recombinant proteins by SDS-PAGE followed by protein staining with the Oriole Fluorescent Gel Stain (Bio-Rad).

HeLa Kyoto cells (1.5 × 10⁵) plated on 60-mm plate were transfected with 5 nM siRNAs. After 6 h incubation, cells were transfected with Halo-ERp44, Ero1α (C99A, C104A)-FLAG, ERAP1-FLAG, or mPrx4-3×FLAG and incubated for additional 36 h. Cells were washed twice and incubated in Opti-MEM for 4 h. Conditioned media were collected and clarified by centrifugation. To concentrate the secreted Halo-ERp44 or mPrx4-3×FLAG proteins, aliquots of resultant supernatants were precipitated by mixing with the same volume of ice-cold 10% TCA. Precipitants were washed with acetone and dissolved in 1 × SDS-loading buffer. To concentrate FLAG-tagged ERp44-client proteins, clarified conditioned media were incubated with anti DYKDDDDK tag antibody beads (Fujifilm Wako Pure Chemicals, 018-22783) at 4 °C with rotation for 3.5 h. Beads were then washed with PBS-T three times, and precipitated proteins were eluted with 1x SDS-loading buffer. To analyze intracellular proteins, cells remaining on plates were lysed in 1 × SDS-loading buffer and homogenized by sonication. Samples were denatured at 70 °C for 10 min, and subjected to SDS-PAGE under reducing conditions. Protein bands were visualized by immunoblotting analyses and quantified by Image Lab software.

Cells that had been cultured in 100-mm dishes with 1 μg/ml of Dox for 24 h were lysed with 550 μl of ice-cold extraction buffer (40 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 1% (w/v) Lubrol, 1 mM DTT, and 0.5 mM AEBSF). The cell lysates were centrifuged, and the supernatants were incubated with anti-DYKDDDDK tag antibody beads (10 μl bed volume, Wako) at 4 °C for 15 min. The beads were washed twice with ice-cold wash buffer 1 (40 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1% (w/v) Lubrol, and 1 mM DTT) and once with ice-cold wash buffer 2 (30 mM potassium phosphate buffer, pH 7.0, 100 mM NaCl, 1 mM MgCl₂, and 0.1% (w/v) Lubrol). Flag-tagged small GTPases were eluted with 60 μl of an acidic solution (100 mM potassium phosphate buffer, pH 3.0, and 0.1% (w/v) Lubrol). The elution (50 μl) was neutralized with 11.2 μl of 0.5 M K₂HPO₄ and heat-denatured at 90 °C for 3 min, to release the guanine nucleotides from small GTPases. The denatured sample (46.2 μl) was mixed with 113.8 μl of distilled water and 1.6 μl of 1 M TBA-B, followed by filtration using an Amicon Ultra 30-kDa centrifugal filter (Merck Millipore). The flow-through fraction was then subjected to IP-RP-HPLC analysis.