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. 2014 Oct 30;10(11):2021–2035. doi: 10.4161/auto.32229

Figure 4.

Figure 4.

NSC185058 effectively inhibits ATG4B activity and LC3B lipidation. (A) Saos-2 (GFP-LC3B) cells were incubated under fed and starved conditions in the presence of 3-MA (10 mM), NSC185058 (100 μM), or NSC377071 (100 μM) for 2, 4, or 6 h. Nonlipidated (GFP-LC3B-I) and lipidated (GFP-LC3B-II) forms of GFP-LC3B were separated by SDS-PAGE and identified by western blotting using anti-GFP (Sigma) antibodies. The band density was quantified and the ratios calculated. (B) The cleavage of LC3B-GST by purified ATG4B was assayed as described in Materials and Methods. (C) The concentration-dependent effect of NSC185058 on ATG4B activity as shown in panel B was quantified. The densities of the LC3B-GST, GST and LC3B bands were measured. The fraction of (GST+LC3B)/(LC3B-GST+GST+LC3B) at each time point is calculated, which correlated with the fraction of products (Fp). Using Fp at time zero (Fp-0) as the baseline, the inhibition% at each time point is calculated as [1-(Fp-t/Fp-0)] × 100%.