Fig. 3: TM9SF4 NTF directly binds to actin to induce actin oxidation.
From: TM9SF4 is an F-actin disassembly factor that promotes tumor progression and metastasis
a Schematic representation of TM9SF4 NTF constructs. TM9SF4 NTF contains a putative copper binding center (purple) and an actin binding domain (ABD, pale yellow). b Co-immunoprecipitation analysis of the binding activity in the region of ABD. NIH3T3 cells were transfected with different constructs, followed by immunoprecipitation using GFP-Trap and analyzed by immunoblotting with anti-actin and anti-GFP antibodies. TM9SF4_N1–177-GFP and TM9SF4_N1–258 (LK220/AA)-GFP failed to immunoprecipitate the actin. c Different GST-tagged TM9SF4 NTFs (2 μM) were incubated with F-actin (2.5 μM), followed by co-sedimentation analysis. S stands for the supernatant fraction, and P stands for pellet fraction. The co-sedimented proteins can be observed in the pellet fraction. d Actin co-sedimentation assay showing that GST-TM9SF4_N24–258(LK220/AA) failed to co-sediment with F-actin. e, f NADH consumption as measured by the decrease of absorbency at 340 nm. In e, different GST-tagged TM9SF4 NTFs (2 μM) were incubated with NADH (400 μM), followed by optical measurement. In f, GST-TM9SF4_N24–258 was treated with or without bathocuproine (25 μM), with or without unfolding by TFA and refolding. g, h G-actin dimer formation after incubation of G-actin (4.2 μM) with GST-TM9SF4_N24–258 (2.5 μM) for 1 h. The samples were treated with or without 5% 2-ME as reducing (R) and non-reducing conditions (NR). N-ethylmaleimide (NEM, 2 mM) was used to block Cys374 of actin. i F-actin glutathionylation after incubation of F-actin (2.5 μM) with GST-TM9SF4_N24–258 (1 μM) in the presence of 10 mM GSH. The samples were resolved in SDS-PAGE and immunoblotted with anti-GSH antibody. j Subtilisin digestion assay showing that GST-TM9SF4_N24–258 treatment reduced the subtilisin digestion of F-actin. Shown are representative immunoblot images detected with anti-actin antibody (left) and summary data (right). Arrow head, cleaved 35 kDa actin fragment. Data are presented as mean ± SEM from 3 biologically independent experiments and two-tailed unpaired Student’s t-test was used for statistical analysis. Source data are provided as a Source Data file.
