2.7. NX1 expression and purification

The rProtein A Sepharose Fastflow Kits (GE Healthcare) was used to purify soluble IgG‐NX1β fusion protein. HEK293 cells were transfected with plasmid expressing the IgG‐NX1β fusion protein, using Lipofectamine 2000. After 72 hr, the supernatant was collected, and protease inhibitors (pepststin,leupeptin,aprotinin and PMSF, a final concentrati of 0.1 g/L; Sigma, US, Santa Clara, California) plus 1 mol/L HEPES‐NaOH were added. Insoluble material was removed by centrifugation (2,500g for 20 min), and the supernatant was retained for the purification assay. Each binding assay used 10 μl IgG beads. The beads were washed three times with 1 ml 1× PBS (with protease inhibitors) by centrifugation (6,500g for 5 min). Then the supernatant was mixed with the beads and rotated at 4°C for at least 6 hr. The supernatant was removed by centrifugation (6,500g for 20 min at 4°C) and the beads washed 3–5 times in 1× PBS (with protease inhibitors). Then elution buffer (glycine, 100 mmol/L) was added and the tubes rotated at room temperature for 30 min. The IgG beads were removed by centrifugation (6,500g for 5 min at 4°C). Neutralizing buffer (2 mol/L Tris‐HCl, pH 8.0) was added to the supernatant and it was saved for immunoprecipitation (IP). Western blot gray scale of triplicate experiments was performed by the software Image J (Clontech Laboratories, Inc. Cat# 632381 RRID:AB_2313808; Jackson ImmunoResearch Labs Cat# 109‐165‐088 RRID:AB_2337725; Jackson ImmunoResearch Labs Cat# 115‐005‐062 RRID:AB_2338452).