FIG. 2.

Transport of the control VSV G tsO45 glycoprotein at different temperatures. 143B cells were transfected with GFP-tagged VSV G tsO45 and incubated at 39°C (nonpermissive temperature) for 24 h and examined immediately by immunofluorescence (A) or following a further 2 h at 20°C (B) or 31°C (D). (C) The cells were incubated at 39°C for 24 h and then at 20°C for 2 h and finally chasedat 31°C for another 2 h. The cells were fixed and stained for TGN46 (in red) (B to D) or with calnexin (an ER marker, also in red) (A). As expected, VSV G tsO45 was transported to the TGN at 20°C (B) and could reach the plasma membrane at 31°C (D, arrows). Upon release of the 20°C block, the block was reversed, and the protein reached the cell surface (C, arrows). Note that under these conditions, only a portion of VSV G tsO45 travels to the plasma membrane at the permissive temperature, as widely reported in the literature.