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. 2005 Jun 21;389(Pt 1):161–172. doi: 10.1042/BJ20041128

Figure 4. ProTGFα N-terminal shedding occurs outside the ER.

Figure 4

(A) Action of brefeldin A on proTGFα forms. CHOTGFα cells were incubated overnight with brefeldin A (10 μg/ml), and/or BB3103 (10 μM). Where indicated, proteinase K was added to the cells (200 μg/ml) for 30 min at room temperature. The different forms of proTGFα were analysed by Western blotting. The arrow indicates the position of the ≈20 kDa form. (B) CHO cells expressing HA-tagged proTGFα were labelled with 500 μCi/ml of [35S]cysteine for 20 min and then chased in complete medium for 30 min. Cells were then washed with cold PBS and incubated at 4 °C with 10 μg/ml of anti-HA for 1 h. Unbound anti-HA antibodies were washed, and the monolayers were incubated in DMEM at 37 °C for the indicated time points. Immune complexes were recovered and analysed by SDS/PAGE, followed by autoradiography. (C) CHOTGFα cells were incubated overnight with brefeldin A (10 μg/ml), and then chased from this compound for the indicated times. ProTGFα forms were analysed by Western blotting. Molecular masses are indicated in kDa in all panels.