Figure 3.

Identification of TPST from bovine adrenal medulla by MSC labeling. Extracts of carbonate-treated membranes solubilized according to protocol 1 (lanes 1–4), 2 (lanes 5 and 6), or 3 (lanes 7–10) were incubated at 0°C for 30 min (lanes 5 and 6) or 1 h (lanes 1–4, 7–10) with bzbz-SgI₃ (lanes 1–4) or bzbz-SgI₃-HA-biotin (lanes 5–10) and [³⁵S]PAPS without (lanes 1, 3, and 5) or with (lanes 2, 4, and 6–10) UV-irradiation, followed by further incubation either for 10 min at 0°C (0°C) or 30°C (30°C) (lanes 1–4) or for 1 h at 37°C (lanes 5 and 6) or 30°C (lanes 7–10) in the absence of UV irradiation. Samples were subjected to SDS/PAGE under reducing (lanes 1–4, 8, and 10) or nonreducing (lanes 5–7, and 9) conditions followed by transfer to poly(vinylidene difluoride) membrane and autoradiography (AR, lanes 1–8). The proteins of lanes 7 and 8 then were immunolabeled for crosslinked SgI₃-HA-biotin peptide using the mAb 12CA5 against the HA epitope followed by enhanced chemiluminescence (Blot, lanes 9 and 10). Open arrows and asterisks indicate the 61- to 57-kDa TPST monomer and 122- to 114-kDa TPST dimer, respectively, bearing crosslinked [³⁵S]sulfate-labeled SgI₃ or SgI₃-HA-biotin peptide.