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. 2010 Jul 12;190(1):45–53. doi: 10.1083/jcb.200908105

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© 2010 Bechler et al.

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Figure 1. — PAFAH Ib catalytic subunits are cytosolic Golgi tubulation factors. (A) Peak Golgi tubule–stimulating activity (determined by an in vitro reconstitution assay) cofractionates with PAFAH activity and PAFAH Ib subunits (Western blot) in final bovine brain cytosol (BBC) fractionation steps. (B) Silver stain of final fractions with the minimal components that stimulate Golgi tubules in vitro. The components identified by MALDI TOF-TOF and/or Western blot are labeled (NS = non specific). (C) Coomassie-stained gel and PAFAH activity of purified subunits. (D) EM of negative stain Golgi from in vitro tubulation reconstitution assays incubated with BBC and purified subunits as indicated. Bars, 500 nm. (E) Quantitation of tubulation assay using purified α1 and α2 subunits (7.5 µg/ml), with subthreshold BBC (low BBC, 0.25 mg/ml) compared with saturating BBC (1.5 mg/ml). svPLA₂ = snake venom PLA₂. Results are the percentage of control (BBC), n = 4; error bars = SD. (F) Anti-α1 and/or anti-α2 antibodies, but not preimmune IgG (PI-α1, PI-α2), inhibit cytosol-stimulated Golgi membrane tubules in vitro. BSA = bovine serum albumin. Results are percentage of control (BBC), n = 4; error bars = SD. (G) Quantitation of tubulation with purified wild-type or LIS1-binding defective α1 E38D and α2 E39D (7.5 µg/ml), in the presence or absence of low BBC. Results are percentage of control (BBC), n = 3; error bars = SEM.