Fig. 3. Class I ARF proteins specifically affect the AP-4 TGN localization. HeLa cells were transiently transfected with pXS-ARF1-T31N-HA (B–D), pXS-ARF3-T31N-HA (E–G), pXS-ARF4-T31N-HA (H–J), pXS-ARF5-T31N-HA (K–M) or pXS-ARF6-T27N-HA (N–P) and incubated for 24 h. (A) Post-nuclear supernatants were prepared and immunoblotting was performed using an anti-HA antibody. (B–P) After 24 h, cells were fixed and double labeled for immunofluorescence microscopy using the anti-σ4 antibody (Cy3) for (B), (E), (H), (K) and (N) and an anti-HA (Alexa 488) antibody for (C), (F), (I), (L) and (O) to detect AP-4 localization and ARF expression, respectively. Transfected cells are indicated by arrows. Bar (B–P), 5 µm.