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Fig. 5 | Molecular Medicine

Fig. 5

From: Phorbol Esters Affect Multiple Steps in β-Amyloid Precursor Protein Trafficking and Amyloid β-Protein Production

Fig. 5

Immunoprecipitation of β PP s after saponin treatment

(A) Transfected wild-type CHO cells were pulse labeled with 35S-methionine for 10 min without a chase period and treated either with 0.1% or 0.2% saponin at 4°C for 40 min. Immunoprecipitation of full-length βPP was carried out with CT15 antibody from saponin buffer (saponin) or cell lysate (lysate), the latter after solubilization with 1% NP40. Full-length βPP (arrowhead) was readily precipitated from cell lysate but was minimally detectable from the buffer fraction treated with 0.2% saponin and essentially undetectable in the 0.1% saponin buffer. (B) Immunoprecipitations of intracellular and secreted βPPs after PDBu treatment. Transfected CHO cells were labeled by 35S-methionine for 10 min and chased for 20 min with (+) or without (−) PDBu. βPPs was immunoprecipitated from medium (media) and from saponin buffer (saponin) with antibodies B5 and 1736. Levels of immunoprecipitable βPPs were higher in media with both 1736 and B5 antibodies but lower intracellularly when recovered from the saponin buffer, after PDBu treatment. Antibody 1736 consistently precipitates less βPPs than antibody B5, accounting for the lower signal in the 1736 lanes. In low-percentage gels, βPPs from CHO cells migrates as a doublet.

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