Characterization of SERPINB6 expressed by HMC-1 cells. (A) Ten microliters HMC-1 cell lysate (4 × 10⁶ cells) was separated on 10% SDS-PAGE and subjected to Western blotting with mAb PI6-25 (1.5 μg/mL) to detect PI6 (lane 1). As positive control, rSERPINB6 was included (lane 2). The position of the molecular mass marker bands is indicated on the left. (B) One microliter HMC-1 lysate (40 × 10⁶ cells) was applied to an ACA-54 column and was eluted with either PBS (black line) or PBS/2 M NaCl (gray dotted line) at 0.5 mL/min. SERPINB6 was measured in the column fractions by SERPINB6 ELISA. Positions of the markers albumin and IgG are indicated for both buffers. (C) HMC-1 lysate and the peak fractions of SERPINB6 of the ACA54 column were immunoprecipitated with pAb anti-SERPINB6 coupled to Sepharose beads and were analyzed on Western blot using mAb PI6-25 or mAb AA1 (1:200) to detect SERPINB6 (lanes 1-2, 5-6) and β-tryptase (lanes 3-4, 7-8), respectively. (arrow, lane 7) 32 kDa β-tryptase. (arrowhead, lanes 6, 8) 78 kDa SERPINB6-β-tryptase complex. The position of the molecular mass marker band is indicated on the left.