8496s capzα1 ms proteintech 66066 1 ig capzβ rb sigma aldrich ab6017 gapdh  (Proteintech)

Journal: bioRxiv

Article Title: Naa10 regulates hippocampal neurite outgrowth via Btbd3 N-α-acetylation-mediated actin dynamics

doi: 10.1101/2024.05.09.583166

Figure Lengend Snippet: (A) Btbd3-interacting proteins are enriched in cytoskeleton proteins. Left: A silver staining gel of total lysates of HT-22 cells with or without expressing Btbd3-Flag subjected to immunoprecipitation (IP) with Flag beads, followed by SDS-PAGE. In-gel digestion was then performed, followed by mass spectrometry analysis. Right: Molecular function analysis of Btbd3-interacting proteins analyzed using the online tool THE GENE ONTOLOGY RESOURCE. (B) N-acetylated Btbd3-interacting proteins. Pie charts show proteins associated with Btbd3 depending on Naa10 and Btbd3 N-acetylation. Flag-bead pull-down experiments in WT HT-22 cells expressing Btbd3 WT -Flag or Btbd3 V1P -Flag or in Naa10-KO HT-22 cells expressing Btbd3 WT -Flag were performed. The list of proteins in each group is shown in Table S3. Bottom: Molecular function analysis of 13 N-acetylated Btbd3-interacting proteins analyzed using the online tool THE GENE ONTOLOGY RESOURCE. (C) Btbd3 binding to CapZb and β-actin depends on Naa10 and Btbd3 N-α-acetylation. Btbd3-Flag IP was performed in WT or Naa10-KO HT-22 cells expressing the indicated proteins, followed by western blotting. Input comprises 10% of the total cell lysates. (D) Naa10 KO reduces the association of CapZb and F-actin. (E) Acetyltransferase activity of Naa10 promotes CapZb association with F-actin. (F) Btbd3 N-α-acetylation enhances CapZb association with F-actin. (D-F) Total cell lysates of WT, Naa10-KO or Btbd3-KO HT-22 cells expressing the indicated proteins were separated into globular actin (G-actin) and F-actin fractions, followed by western blotting. The bands of CapZb and β-actin in F-actin fraction were quantified using ImageJ. The relative ratios of CapZb to β-actin of each lane compared to lanes 3 (D) , 4 (E) and 5 (F) are shown. (C-F) Repeats of western blotting experiments from figure 3.

Article Snippet: For western blotting, the following Abs were used: mouse monoclonal anti-Naa10 Ab (sc-373920, Santa Cruz), mouse monoclonal anti-actin Ab (MAB1501, Merck Millipore), anti-α-tubulin Ab (T5168, Sigma), mouse monoclonal anti-Flag (M2) (F3165, Sigma), mouse monoclonal anti-Btbd3 Ab (TA808669S, OriGene), and mouse monoclonal anti-CapZb Ab (sc-136502, Santa Cruz).

Techniques: Silver Staining, Expressing, Immunoprecipitation, SDS Page, Mass Spectrometry, Binding Assay, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Naa10 regulates hippocampal neurite outgrowth via Btbd3 N-α-acetylation-mediated actin dynamics

doi: 10.1101/2024.05.09.583166

Figure Lengend Snippet: (A) Btbd3 binding to CapZb and β-actin depends on Naa10 and Btbd3 N-α-acetylation. Btbd3-Flag immunoprecipitation (IP) was performed in WT or Naa10-KO HT-22 neurons expressing the indicated proteins, followed by western blotting using antibodies against Flag, CapZb, β-actin, and Naa10. Input represents 10% of total cell lysates. The experiment was replicated twice, with the additional replicate shown in . (B) Naa10 KO reduces the association of CapZb and F-actin. (C) Acetyltransferase activity of Naa10 promotes CapZb association with F-actin. (D) Btbd3 N-α-acetylation enhances CapZb association with F-actin. Western blotting was performed using Abs against CapZb, β-actin, and Btbd3. (B-D) Total cell lysates of WT, Naa10-KO or Btbd3-KO HT-22 neurons expressing the indicated proteins were separated into globular actin (G-actin) and F-actin fractions, followed by western blotting. The bands of CapZb and β-actin in F-actin fraction were quantified using ImageJ. The relative ratios of CapZb to β-actin of each lane compared to lanes 3 (B) , 4 (C) and 5 (D) are shown. The bar charts display the relative ratios of CapZb to β-actin in the F-actin fraction with individual data points and mean ± SD for each group from three independent experiments (see -F for replicates). (B) analyzed by unpaired two-tailed t test. (C-D) analyzed by two-way ANOVA plus Sidak’s post-hoc.

Article Snippet: For western blotting, the following Abs were used: mouse monoclonal anti-Naa10 Ab (sc-373920, Santa Cruz), mouse monoclonal anti-actin Ab (MAB1501, Merck Millipore), anti-α-tubulin Ab (T5168, Sigma), mouse monoclonal anti-Flag (M2) (F3165, Sigma), mouse monoclonal anti-Btbd3 Ab (TA808669S, OriGene), and mouse monoclonal anti-CapZb Ab (sc-136502, Santa Cruz).

Techniques: Binding Assay, Immunoprecipitation, Expressing, Western Blot, Activity Assay, Two Tailed Test