Figure 1
Association of serglycin with fully processed HNP. Immunoplates were incubated with affinity purified Ab. Plates were washed, blocked, and incubated with wild-type or transfected 32D Cl3 cell lysates. (A-B) To assess the amount of serglycin and HNP-1 bound by the anti-serglycin Ab in the mixed ELISA, serglycin and HNP-1 was measured by sandwich ELISA in lysates of 32D Cl3 cells and 32D Cl3 cells stably transfected with proHNP (32D Cl3+proHNP) before and after catching with anti-serglycin Ab. (C-G) Lysates of 32D Cl3 cells were subjected to a mixed ELISA. Serglycin was captured with affinity purified anti-serglycin Ab. Association of a protein with serglycin was probed with a goat or biotinylated rabbit Ab. After incubation with HRP-conjugated rabbit anti–goat Ab or HRP-conjugated avidin, a color reaction was developed using OPD tablets. Color reaction was measured at an absorbance (Abs) of 492 nm and background reactivity subtracted (absorbance in wells with lysis buffer without cells). Absorbance points to an association of a protein with serglycin. (C) Mixed ELISA on lysates of 32D Cl3 cells and 32D Cl3+proHNP using a biotinylated Ab against proHNP, which does not recognize fully processed HNP-1, and a biotinylated Ab against mature HNPs which recognizes fully processed HNP-1. (D) Previous ELISA was repeated, but with cABC treatment as an extra step after incubation with cell lysates. Note that cABC treatment abolished reactivity for the cationic HNP-1 indicating that reactivity is dependent on serglycin's anionic chondroitin sulfate sidechains. (E) Other potential binding candidates of serglycin were tested on a mixed ELISA on lysates of 32D Cl3 cells and 32D Cl3+proHNP using Abs against murine myeloperoxidase (MPO), neutrophil elastase (mNE), and proteinase 3 (mPR3). (F) 32D Cl3+proHNP cells were further transfected with human neutrophil elastase (hNE) or proteinase 3 (hPR3) to test whether signal could be enhanced by greater expression. Probing was done with Abs raised against hNE or hPR3. (G) Serglycin HNP-1 association was probed by mixed ELISA in 32D Cl3 cells transfected with HNP-1 and hNE or hPR3 to test whether high expression of hNE could displace HNP-1.

Association of serglycin with fully processed HNP. Immunoplates were incubated with affinity purified Ab. Plates were washed, blocked, and incubated with wild-type or transfected 32D Cl3 cell lysates. (A-B) To assess the amount of serglycin and HNP-1 bound by the anti-serglycin Ab in the mixed ELISA, serglycin and HNP-1 was measured by sandwich ELISA in lysates of 32D Cl3 cells and 32D Cl3 cells stably transfected with proHNP (32D Cl3+proHNP) before and after catching with anti-serglycin Ab. (C-G) Lysates of 32D Cl3 cells were subjected to a mixed ELISA. Serglycin was captured with affinity purified anti-serglycin Ab. Association of a protein with serglycin was probed with a goat or biotinylated rabbit Ab. After incubation with HRP-conjugated rabbit anti–goat Ab or HRP-conjugated avidin, a color reaction was developed using OPD tablets. Color reaction was measured at an absorbance (Abs) of 492 nm and background reactivity subtracted (absorbance in wells with lysis buffer without cells). Absorbance points to an association of a protein with serglycin. (C) Mixed ELISA on lysates of 32D Cl3 cells and 32D Cl3+proHNP using a biotinylated Ab against proHNP, which does not recognize fully processed HNP-1, and a biotinylated Ab against mature HNPs which recognizes fully processed HNP-1. (D) Previous ELISA was repeated, but with cABC treatment as an extra step after incubation with cell lysates. Note that cABC treatment abolished reactivity for the cationic HNP-1 indicating that reactivity is dependent on serglycin's anionic chondroitin sulfate sidechains. (E) Other potential binding candidates of serglycin were tested on a mixed ELISA on lysates of 32D Cl3 cells and 32D Cl3+proHNP using Abs against murine myeloperoxidase (MPO), neutrophil elastase (mNE), and proteinase 3 (mPR3). (F) 32D Cl3+proHNP cells were further transfected with human neutrophil elastase (hNE) or proteinase 3 (hPR3) to test whether signal could be enhanced by greater expression. Probing was done with Abs raised against hNE or hPR3. (G) Serglycin HNP-1 association was probed by mixed ELISA in 32D Cl3 cells transfected with HNP-1 and hNE or hPR3 to test whether high expression of hNE could displace HNP-1.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal