bms1  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.

doi: 10.1016/j.celrep.2024.113891

Figure Lengend Snippet: Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or BMS1 (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Antibodies used for immunoprecipitation were FLAG (ab205606, Abcam), BMS1 (sc-271040, Santa Cruz), FBL (ab226178, Abcam).

Techniques: Staining, RNA Immunoprecipitation, Binding Assay, Two Tailed Test

Journal: Cell reports

Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.

doi: 10.1016/j.celrep.2024.113891

Figure Lengend Snippet: Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or BMS1 (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Samples were separated using 10% acrylamide/bis solution 37.5:1 (Biorad) gels and analyzed by immunoblotting using antibodies against FLAG (Sigma), RNA Pol I (Cell Signaling or Invitrogen), NPM1 (Sigma), FCF1 (Invitrogen), VCL (Sigma), BMS1 (Bethyl), FBL (Abcam), NOP56 (Bethyl), V5 (Invitrogen), RPS6 (Cell Signaling).

Techniques: Staining, RNA Immunoprecipitation, Binding Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Modulation of tau tubulin kinases (TTBK1 and TTBK2) impacts ciliogenesis

doi: 10.1038/s41598-023-32854-4

Figure Lengend Snippet: Structures and data for published TTBK inhibitors. ( A ) Structures of published TTBK1/2 inhibitors and PDB codes for corresponding structures. ( B ) AMG28 MRC panel screening results for kinases with < 15% activity remaining. IDG kinases indicated using asterisks and orange bars.

Article Snippet: Co-crystal structures with TTBK1 have been deposited for inhibitors discovered by AstraZeneca, Bristol-Myers Squibb, and Biogen (AZ1, AZ2, BMS1, and BGN18, Fig. A) .

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Modulation of tau tubulin kinases (TTBK1 and TTBK2) impacts ciliogenesis

doi: 10.1038/s41598-023-32854-4

Figure Lengend Snippet: X-ray crystallographic structures of human TTBK1 in complex with several analogs and corresponding thermal shift data for TTBK1/2. ( A ) AMG28 (PDB code: 7ZHN) is shown in brown, 10 (PDB code: 7ZHQ) in yellow, and 9 (PDB code: 7ZHP) in pink stick representation, respectively. Image created using Schrödinger PyMOL 2.5. ( B ) 3 (PDB code: 7ZHO) is shown in pink. The hinge region and αC helix are labeled in each panel and hydrogen bonds are included as black dashed lines. Image created using Schrödinger PyMOL 2.5. ( C ) Table of thermal shift (ΔTm) data for TTBK inhibitors.

Article Snippet: Co-crystal structures with TTBK1 have been deposited for inhibitors discovered by AstraZeneca, Bristol-Myers Squibb, and Biogen (AZ1, AZ2, BMS1, and BGN18, Fig. A) .

Techniques: Labeling

Journal: Scientific Reports

Article Title: Modulation of tau tubulin kinases (TTBK1 and TTBK2) impacts ciliogenesis

doi: 10.1038/s41598-023-32854-4

Figure Lengend Snippet: Cell-based NanoBRET assays with TTBK lead inhibitors. ( A ) Structure of tracer 29 . ( B ) Competition experiments between tracer 29 and analog 3 , 9 , or 10 at either 10 or 30 µM for NLuc-TTBK1 and NLuc-TBK2, n = 1. ( C ) Tracer titration results for NLuc-TTBK1 and NLuc-TTBK2 in the presence of analog 3 , n = 1. Calculated IC 50 values for 3 are shown for each kinase at the recommended working tracer concentration of 2 µM. ( D ) Representative NanoBRET assay curves and corresponding IC 50 values for active compounds AMG28, 3 , 9 , and 10 in intact HEK293 cells, n = 2. Error is shown and reported as standard error mean (SEM).

Article Snippet: Co-crystal structures with TTBK1 have been deposited for inhibitors discovered by AstraZeneca, Bristol-Myers Squibb, and Biogen (AZ1, AZ2, BMS1, and BGN18, Fig. A) .

Techniques: Titration, Concentration Assay

Journal: Scientific Reports

Article Title: Modulation of tau tubulin kinases (TTBK1 and TTBK2) impacts ciliogenesis

doi: 10.1038/s41598-023-32854-4

Figure Lengend Snippet: Effects of TTBK lead inhibitors on downstream signaling. ( A ) Western blot analyses of SH-SY5Y cells after 48 h treatment with 9 or 10 . Quantification of phospho-TDP-43 (Ser409/410, normalized to total TDP-43), n = 3. *control versus 5 µM 10 p -value = 0.0017. ( B ) Western blot analyses of SH-SY5Y cells after 48 h treatment with 9 or 10 . Quantification of total TDP-43, n = 3. *control versus 5 µM 10 p -value = 0.0001. Error bars represent SEM. P -values were generated using one-way ANOVA with Tukey correction. Unformatted images of blots are included in Fig. S8.

Article Snippet: Co-crystal structures with TTBK1 have been deposited for inhibitors discovered by AstraZeneca, Bristol-Myers Squibb, and Biogen (AZ1, AZ2, BMS1, and BGN18, Fig. A) .

Techniques: Western Blot, Control, Generated