Fig 2: KATNA1 interacts with SUMO proteins.A, identification of KATNA1-binding proteins. Lysates from rat brain tissues were subjected to pulldown with GST or GST-KATNA1. Precipitated proteins were subjected to SDS-PAGE followed by staining with Fast Silver Stain Kit. , tandem mass spectrometry spectrum of a UBC9 peptide identified from pulldown sediments using GST-KATNA1 fusion protein. C and D, GST pulldown assays were performed with rat brain lysates. GST plasmids were constructed and proteins were purified (lower panel; the arrow points to the target protein). E, GFP-KATNA1 and HA-SUMO2 were overexpressed in 293T cells with Flag-UBC9. Coimmunoprecipitation with an anti-HA antibody was used to isolate proteins from the cell lysates, and the proteins were visualized using Western blotting and detected with anti-GFP and anti-HA antibodies. F, proteins were immunoprecipitated from rat brain lysates with a KATNA1 antibody, and the precipitates were immunostained with a SUMO2 antibody after Western blotting. G, immunocytochemistry for SUMO2 (green) and KATNA1 (red) was performed in hippocampal neurons at DIV3. White arrows indicate colocalization (yellow in merged image) in the neurite. The scale bar represents 20 µm. The white rectangle indicates the region chosen to show enlarged details (lower panel, the scale bar represents 10 µm). H, the fluorescence intensity profiles for KATNA1 and SUMO2 from the white line in the lower panel of (G).

Fig 3: SUMOylation of KATNA1 enhances hippocampal neurite outgrowth. Hippocampal neurons were transfected with HA-UBC9 and GFP-KATNA1, or the K330R mutant (GFP vector as control), with or without Flag-SUMO2 (Flag vector as control) for 48 h. Neurons were stained with immunofluorescent antibodies for GFP (green), Flag (purple), and MAP-2 (red for dendrite). The white dashed line indicates the edge where two images were merged. The scale bar represents 100 µm.

Fig 4: SUMOylation of KATNA1 promotes the microtubule severing activity of KATNA1.A, COS7 cells were transfected with different GFP-KATNA1 constructs with or without plasmids encoding mCherry-SUMO2; GFP or mCherry vector was used as control. Cells were stained with immunofluorescent antibodies for GFP (green), mCherry (red), and tubulin (white). GFP fluorescence indicated the successfully transfected cells, circled with white line. Nuclei were stained with DAPI (blue). The scale bar represents 20 µm. B, the relative fluorescence intensity of tubulin in COS7 cells was quantified. (n = 3; n corresponds to the number of independent experiments, cell number > 20/group for each replicate). Values represent mean ± SD; the data were analyzed with one-way analysis of variance (ANOVA), ****p < 0.0001.