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. 2011 Jul 15;124(14):2401–2413. doi: 10.1242/jcs.083782

Fig. 9.

Fig. 9.

Increasing the length of the transmembrane domain of TGN38 reduces the efficiency of Golgi retrieval from early endosomes. HeLa cells were transfected with full-length TGN38 (wtTGN38) or TGN38TM+3 for 24 hours. Monolayers were incubated with mouse or rabbit anti-TGN38 antibodies for 30 minutes on ice, washed in PBS and incubated in serum-free medium for 120 minutes at 37°C and then fixed and permeabilised. (A,B) The internalised antibody-bound TGN38 or TGN38TM+3 was stained with (A) Alexa-Fluor-568-conjugated anti-mouse IgG (red) and p230 using rabbit polyclonal anti-p230 antibodies (green) or (B) Alexa-Fluor-568-conjugated anti-rabbit IgG (red) and EEA1 using mouse monoclonal anti-EEA1 (green). (C) Quantification of TGN38TM+3 levels at the Golgi. The percentage of the total TGN38 pixels that overlapped with p230 in each cell after 120 minutes of internalisation was determined using the plug-in OBCOL on ImageJ wtTGN38. Data are expressed as a percentage (±s.e.m.) of wtTGN38 (n=35 for each sample). ***P<0.001. Scale bars: 10 μm.