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. 2021 Nov 8;221(1):e202106100. doi: 10.1083/jcb.202106100

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© 2021 Pennauer et al.

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Figure 7. — Rescue experiments of the Arf4ko phenotype by overexpressing Arf1, Arf4, or Arf5. Stable cell lines were generated from parental HeLaα and Arf4ko cells by lentiviral transduction, either expressing the empty vector (+empty) as a control or overexpressing the indicated untagged Arf. (A and B) Expression levels of Arfs in clonal (A) and pooled (B) cell lines were analyzed by immunoblotting. Ten and five times less lysate were loaded for Arf-overexpressing cell lines in A and B, respectively. In B, two irrelevant lanes were removed as indicated by the black line. Actin served as a loading control. (C and D) Immunoblot analysis probing the media (M) and cell lysates (L) for the chaperones BiP and calreticulin (Calret.) of parental HeLaα and Arf4ko cell lines transduced with the empty vector or overexpressing class II Arfs (C) or class I Arf Arf1 (D). Actin served as a loading control. (E and F) Immunofluorescent microscopy revealed the steady-state localization of transiently expressed signal sequence–GFP–KDEL (green) coimmunostained with GM130 (magenta) in parental HeLaα and Arf4ko cell lines transduced with the empty vector or overexpressing class II Arfs Arf4 or Arf5 (E) or overexpressing class I Arf Arf1 (F). DAPI signal is shown in blue. Lower panels show magnified image sections. Asterisks mark images with enhanced intensity. Scale bars, 10 µm.