Journal: bioRxiv

Article Title: Physiological and pathophysiological homeostasis of astroglial channel proteins by Nedd4-2

doi: 10.1101/683441

Figure Lengend Snippet: (A) Time course of ubiquitination using recombinant wild type Nedd4-2 (Nedd4-2 WT). Purified Ubi was incubated with ATP, E1, E2, and Nedd4-2 WT for indicated durations. Samples were subjected for Western blotting using anti-Ubi (top panel) and anti-Nedd4-2 (bottom panel) antibodies. Note the time-dependent polyUbi chain formation at the expense of free Ubi (arrowhead). (B) The average time course of the depletion of free Ubi (left) and formation of polyUbi chains (right) in in vitro ubiquitination assay in (A) (n=3 replicates). (C to F) S233L (C and D) and H515P (E and F) missense point mutants of Nedd4-2 cause gain-of-function of the catalytic activity. (C and E) Representative images of Western blotting using anti-Ubi (upper panel) and anti-Nedd4-2 (lower panel) antibodies for in vitro ubiquitination assay samples using Nedd4-2 WT and mutants [S233L in (C) and H515P in (E) ]. Arrowheads, free Ubi. (D and F) Quantifications of depletion of free Ubi (top dot plots) and formation of polyUbi chains relative to N4-2 WT (bottom dot plots) after 150 sec incubation. Nedd4-2 WT (black dots) and Nedd4-2 mutants (magenta dots). Results are shown as mean ± SEM. Numbers of experiments are 4 for each set of experiments. ***, p <0.001; **, 0.001< p <0.01; *, 0.01< p <0.05 (Student’s t-test). See also Table S2.

Article Snippet: For in vitro ubiquitination assays, recombinant mouse E1 (Addgene plasmid # 32534), E2 (pGEX4T-1-UbcH5b), and E3s (pDEST-Nedd4-2) were expressed in BL21 Rosetta 2 (DE3) E.coli and purified according to published protocols ( ; ; ) with slight modifications.

Techniques: Ubiquitin Proteomics, Recombinant, Purification, Incubation, Western Blot, In Vitro, Activity Assay

Journal: bioRxiv

Article Title: Nuclear growth and import can be uncoupled

doi: 10.1101/2023.04.19.537556

Figure Lengend Snippet: X. laevis egg extracts were supplemented with 1 µM Set9, 100 µM Zebularine, or an equivalent volume of appropriate buffer (Control). X. laevis sperm chromatin was then added to induce nuclear assembly. Nuclei were fixed and stained for the NPC (mAb414) or DNA (Hoechst or EthD-2). (A) Thresholding was applied to a representative nucleus stained for DNA or NPCs (red). The thresholded DNA area was divided by the thresholded total nuclear CS area to calculate a parameter we refer to as the “relative chromatin area.” Higher values represent less-compact chromatin, whereas lower values represent more-compact chromatin. Scale bar, 5 µm. (B) Representative images of Hoechst-stained nuclei are shown. Scale bar, 5 µm. (C) Representative images of NPC-stained nuclei are shown. Scale bar, 5 µm. (D) Relative chromatin area was quantified for 73 control nuclei and 59 Set9 nuclei from three independent experiments. (E) Nuclear CS area was quantified based on NPC staining for 919 control nuclei and 1177 Set9 nuclei from five independent experiments. (F) Representative images of EthD-2–stained nuclei are shown. Scale bar, 5 µm. (G) Representative images of NPC-stained nuclei are shown. Scale bar, 5 µm. (H) Relative chromatin area was quantified for 81 control nuclei and 58 Zebularine-treated nuclei from two independent experiments. (I) Nuclear CS area was quantified for 260 control nuclei and 235 Zebularine-treated nuclei from three independent experiments. Two-tailed Student’s t-tests assuming equal variances: ****p < 0.0001. Error bars represent SD.

Article Snippet: His-tagged Set9 protein expression was induced overnight at room temperature with 1 mM IPTG using BL21(DE3) Escherichia coli transformed with pET28 Set9 plasmid (Addgene plasmid #24082).

Techniques: Control, Staining, Two Tailed Test