Journal: Cell Reports

Article Title: The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to prevent spurious loading of illegitimate small RNA

doi: 10.1016/j.celrep.2022.110671

Figure Lengend Snippet:

Article Snippet: Anti cMyc-HRP , Miltenyi , Cat# 130-092-113, RRID: AB_871937.

Techniques: Virus, Recombinant, SYBR Green Assay, Western Blot, Protease Inhibitor, Isolation, Reverse Transcription, Magnetic Beads, Mass Spectrometry, Cloning, Sequencing, Software

Journal: Cancer cell

Article Title: Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis.

doi: 10.1016/j.ccr.2009.03.021

Figure Lengend Snippet: Figure 7. Inhibition of Oncogenes Plays a Significant Role in Apoptosis Induction by Pharmacologically Activated p53 (A) Detection of apoptotic cells by FACS of annexin-stained HCT116 and MCF7 cells after 24 hr of treatment with 0.1 and 1 mM RITA and by phase-contrast microscopy of MCF7 cells. (B and C) Cell death induction was assessed by trypan blue staining of cells treated with a low dose of RITA upon knockdown of c-Myc and Mcl-1 by siRNA in (B) HCT116 and (C) MCF7 cells (mean ± SEM, n = 3). (D) Phase-contrast microscopy of MCF7 cells treated with 0.1 mM RITA upon c-Myc or Mcl-1 knockdown. Scale bars represent 100 mm. (E) Upper panel: levels of IGF1R, c-Myc, Mcl-1, survivin, and p53 in MCF7, U2OS, and HCT116 cells treated with 10 mM nutlin3a were assessed by western blot. Lower panel: cell death induced by nutlin3a in the presence or absence of c-Myc or Mcl-1 depletion by siRNA was detected by try- pan blue staining (mean ± SD, n = 3). (F and G) Cell death of HCT116 cells treated with the indicated combinations of RITA and the PI3- kinase inhibitor LY294002 was assessed by (F) try- pan blue staining (mean ± SD, n = 3) or (G) phase- contrast microscopy. The scale bars in (A), (G), and (D) represent 100 mm. The asterisk in (B), (C), (E), and (F) denotes an expected additive effect.

Article Snippet: MYC siRNA was kindly provided by L.-G. Larsson, MCL-1 siRNA was purchased from Santa Cruz, and GFP siRNA was purchased from Oligoengine.

Techniques: Inhibition, Staining, Microscopy, Knockdown, Western Blot

Journal: eLife

Article Title: A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

doi: 10.7554/elife.79771

Figure Lengend Snippet: Figure 2. Rab8A and Rab10 bind to the LRRK2 Armadillo domain. (A–C) Microscale thermophoresis of labeled, LRRK2 Armadillo domain fragments comprised of residues 1–552, 1–159, or 350–550 with Rab8A Q67L as indicated. (C–E) Microscale thermophoresis for Rab10 Q68L (1–181) with indicated LRRK2 Armadillo fragments, as in (A). Purified Rab proteins were serially diluted and then NHS-RED-labeled LRRK2 Armadillo domain (final concentration 100 nM) was added. Graphs show mean and SEM from three independent measurements, each from a different set of protein preparations. Data are summarized in Table 1.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody Anti- LRRK2 (mouse monoclonal) NeuroMab RRID:AB_2877351 N241A/34 (1:1000) Antibody Anti- LRRK2 phospho S935 (rabbit monoclonal) Abcam RRID:AB_2904231 UDD2 (1:1000) Antibody Anti- Rab10 (mouse monoclonal) Nanotools RRID:AB_2921226 0680- 100/Rab10605B11 (1:1000) Antibody Anti- Rab10 (phospho T73) (rabbit monoclonal) Abcam RRID:AB_2811274 ab230261 (1:1000) Antibody Anti- FLAG M2 (mouse monoclonal) MilliporeSigma RRID:AB_262044 F- 1804 (1:2000) Strain, strain background (Escherichia coli) E. coli DH5α Thermo Fisher 18258012 Strain, strain background (E. coli) E. coli STBL3 Thermo Fisher C737303 Strain, strain background (E. coli) E. coli Rosetta DE3 pLys Millipore 70956 Cell line (Homo sapiens) HeLa ATCC CCL- 2 Cell line (H. sapiens) HEK293T ATCC CRL- 3216 Chemical compound, drug MLi- 2 MRC PPU Chemical compound, drug Creatine phosphate Fluka Analytical #27920 20 mM Commercial assay or kit RED- NHS 2nd Generation (Amine Reactive) Protein Labeling Kit NanoTemper Technologies MO- L011 Commercial assay or kit CF 633 Succinimidyl Ester Protein Labeling Kit Biotium #92217 Other Creatine Phosphokinase Sigma C3755 30U Chemical compound, drug 18:1 (Δ9- Cis) PC (DOPC) Avanti Polar Lipids #850375 11 μmol Chemical compound, drug 18:1 PS (DOPS) Avanti Polar Lipids #840035 5 μmol Chemical compound, drug 18:1 DGS- NTA(Ni) Avanti Polar Lipids #790404 0.85 μmol Chemical compound, drug 18:1 PI(4)P Avanti Polar Lipids #850151 0.15 μmol Chemical compound, drug DiD Thermo Fisher D7757 0.01 μmol Recombinant DNA reagent pNIC Bsa- 4 His- Sumo Rab10 Q68L 1–181 Gift of Amir Khan Human Recombinant DNA reagent pET15b His- Mst3 Gift of Amir Khan Human Recombinant DNA reagent pET21b GFP- Rab10 Q68L- His Addgene RRID:Addgene_186015 186015 Human Recombinant DNA reagent pET21b His Rab8A Q67L Addgene RRID:Addgene_186014 186014 Human Vides et al. eLife 2022;11:e79771.

Techniques: Microscale Thermophoresis, Labeling, Purification, Concentration Assay

Journal: eLife

Article Title: A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

doi: 10.7554/elife.79771

Figure Lengend Snippet: Figure 5. Identification of a basic patch at the N-terminus of LRRK2 that is needed for phosphoRab interaction. (A) Electrostatic surface potential of LRRK2 Armadillo domain residues 1–552 modeled using Chimera 2 software (Pettersen et al., 2004); blue indicates a positively charged surface. LRRK2 K17 and K18 are indicated. (B) AlphaFold (Jumper et al., 2021) structure of putative, active LRRK2 with residues that mediate Rab29 binding shown in red (site #1) and the K17/K18 residues that are required for phosphoRab10 binding (site #2) shown in magenta; the kinase domain is shown in blue. (C, D) Microscale thermophoresis of labeled, full-length LRRK2 K17A or K18A Armadillo 1–552 with His phosphoRab10 Q68L 1–181. Purified Rab10 protein was phosphorylated with Mst3 kinase at 27°C for 2 hr and then serially diluted; NHS-RED-labeled Armadillo (final concentration 100 nM) was then added. Graphs show mean and SEM from three independent measurements, each from a different set of protein preparations.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody Anti- LRRK2 (mouse monoclonal) NeuroMab RRID:AB_2877351 N241A/34 (1:1000) Antibody Anti- LRRK2 phospho S935 (rabbit monoclonal) Abcam RRID:AB_2904231 UDD2 (1:1000) Antibody Anti- Rab10 (mouse monoclonal) Nanotools RRID:AB_2921226 0680- 100/Rab10605B11 (1:1000) Antibody Anti- Rab10 (phospho T73) (rabbit monoclonal) Abcam RRID:AB_2811274 ab230261 (1:1000) Antibody Anti- FLAG M2 (mouse monoclonal) MilliporeSigma RRID:AB_262044 F- 1804 (1:2000) Strain, strain background (Escherichia coli) E. coli DH5α Thermo Fisher 18258012 Strain, strain background (E. coli) E. coli STBL3 Thermo Fisher C737303 Strain, strain background (E. coli) E. coli Rosetta DE3 pLys Millipore 70956 Cell line (Homo sapiens) HeLa ATCC CCL- 2 Cell line (H. sapiens) HEK293T ATCC CRL- 3216 Chemical compound, drug MLi- 2 MRC PPU Chemical compound, drug Creatine phosphate Fluka Analytical #27920 20 mM Commercial assay or kit RED- NHS 2nd Generation (Amine Reactive) Protein Labeling Kit NanoTemper Technologies MO- L011 Commercial assay or kit CF 633 Succinimidyl Ester Protein Labeling Kit Biotium #92217 Other Creatine Phosphokinase Sigma C3755 30U Chemical compound, drug 18:1 (Δ9- Cis) PC (DOPC) Avanti Polar Lipids #850375 11 μmol Chemical compound, drug 18:1 PS (DOPS) Avanti Polar Lipids #840035 5 μmol Chemical compound, drug 18:1 DGS- NTA(Ni) Avanti Polar Lipids #790404 0.85 μmol Chemical compound, drug 18:1 PI(4)P Avanti Polar Lipids #850151 0.15 μmol Chemical compound, drug DiD Thermo Fisher D7757 0.01 μmol Recombinant DNA reagent pNIC Bsa- 4 His- Sumo Rab10 Q68L 1–181 Gift of Amir Khan Human Recombinant DNA reagent pET15b His- Mst3 Gift of Amir Khan Human Recombinant DNA reagent pET21b GFP- Rab10 Q68L- His Addgene RRID:Addgene_186015 186015 Human Recombinant DNA reagent pET21b His Rab8A Q67L Addgene RRID:Addgene_186014 186014 Human Vides et al. eLife 2022;11:e79771.

Techniques: Software, Binding Assay, Microscale Thermophoresis, Labeling, Purification, Concentration Assay

Journal: eLife

Article Title: A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

doi: 10.7554/elife.79771

Figure Lengend Snippet: Figure 8. Feed-forward pathway for Rab10 phosphorylation is dependent on LRRK2 kinase activity. (A) Fluorescence intensity traces of individual, single molecules of 7 nM CF633-labeled FLAG-LRRK2 R1441G on a substrate-supported lipid bilayer decorated with lipid-anchored GFP-Rab10 Q68L-His across 600 s of live total internal reflection (TIRF) microscopy. Red, R1441G; blue, K17A/K18A/R1441G; yellow, D2017A. (B) Reactions were carried out as in (A) except Rab10 was omitted (purple) or Rab10 was replaced with Rab11 (green). Dashed lines in (A) and (B) represent time of addition of fluorescently labeled LRRK2 at 60 s; shown are representative experiments carried out at least three times for each condition. Fluorescence intensity was fitted by a nonlinear regression curve for two-phase association. Fold change was calculated by dividing the average fluorescence intensity at steady state and subtracting background fluorescence intensity average determined from 60 s prior to LRRK2 addition. (C) Rate of membrane association of LRRK2 as a function of Rab10 concentration. This curve was fitted by a nonlinear regression fit using PRISM software (MathWorks) to determine a Hill coefficient. Data are from two independent experiments plotted together.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody Anti- LRRK2 (mouse monoclonal) NeuroMab RRID:AB_2877351 N241A/34 (1:1000) Antibody Anti- LRRK2 phospho S935 (rabbit monoclonal) Abcam RRID:AB_2904231 UDD2 (1:1000) Antibody Anti- Rab10 (mouse monoclonal) Nanotools RRID:AB_2921226 0680- 100/Rab10605B11 (1:1000) Antibody Anti- Rab10 (phospho T73) (rabbit monoclonal) Abcam RRID:AB_2811274 ab230261 (1:1000) Antibody Anti- FLAG M2 (mouse monoclonal) MilliporeSigma RRID:AB_262044 F- 1804 (1:2000) Strain, strain background (Escherichia coli) E. coli DH5α Thermo Fisher 18258012 Strain, strain background (E. coli) E. coli STBL3 Thermo Fisher C737303 Strain, strain background (E. coli) E. coli Rosetta DE3 pLys Millipore 70956 Cell line (Homo sapiens) HeLa ATCC CCL- 2 Cell line (H. sapiens) HEK293T ATCC CRL- 3216 Chemical compound, drug MLi- 2 MRC PPU Chemical compound, drug Creatine phosphate Fluka Analytical #27920 20 mM Commercial assay or kit RED- NHS 2nd Generation (Amine Reactive) Protein Labeling Kit NanoTemper Technologies MO- L011 Commercial assay or kit CF 633 Succinimidyl Ester Protein Labeling Kit Biotium #92217 Other Creatine Phosphokinase Sigma C3755 30U Chemical compound, drug 18:1 (Δ9- Cis) PC (DOPC) Avanti Polar Lipids #850375 11 μmol Chemical compound, drug 18:1 PS (DOPS) Avanti Polar Lipids #840035 5 μmol Chemical compound, drug 18:1 DGS- NTA(Ni) Avanti Polar Lipids #790404 0.85 μmol Chemical compound, drug 18:1 PI(4)P Avanti Polar Lipids #850151 0.15 μmol Chemical compound, drug DiD Thermo Fisher D7757 0.01 μmol Recombinant DNA reagent pNIC Bsa- 4 His- Sumo Rab10 Q68L 1–181 Gift of Amir Khan Human Recombinant DNA reagent pET15b His- Mst3 Gift of Amir Khan Human Recombinant DNA reagent pET21b GFP- Rab10 Q68L- His Addgene RRID:Addgene_186015 186015 Human Recombinant DNA reagent pET21b His Rab8A Q67L Addgene RRID:Addgene_186014 186014 Human Vides et al. eLife 2022;11:e79771.

Techniques: Phospho-proteomics, Activity Assay, Fluorescence, Labeling, Microscopy, Membrane, Concentration Assay, Software

Journal: eLife

Article Title: A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

doi: 10.7554/elife.79771

Figure Lengend Snippet: Figure 9. PhosphoRab8A activates LRRK2 phosphorylation of Rab10 in solution. (A) Immunoblot analysis of the kinetics of LRRK2 G2019S phosphorylation of Rab10 with and without additional pRab8. Upper gel: GFP-Rab10 Q68L His substrate. Lower gel: His-Sumo-Rab10 wild-type full-length substrate. Indicated reactions contained 200 nM MLi-2. pRab8A was detected with anti-phosphoRab8A antibody. (B) Same as panel (A) with K18A-LRRK2-R1441G and His-Sumo-Rab10 wild-type full-length as substrate. PhosphoRab8A was detected with total Rab8 antibody. (C) Kinetics of phosphoRab10 production as in (A). Shown are the combined means of independent, quadruplicate determinations ± SEM, as indicated. (D) PhosphoRab10 production as in (B). Shown are the combined means of independent duplicate determinations,± SEM, as indicated. Background signal in the presence of pRab8A is likely due to trace MST3 contamination that is not sensitive to MLi-2 inhibition and was subtracted. pRab8 preparation was by method #1 for (A), upper gel, and (B), and method #2 was used in panel (A), lower gel.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody Anti- LRRK2 (mouse monoclonal) NeuroMab RRID:AB_2877351 N241A/34 (1:1000) Antibody Anti- LRRK2 phospho S935 (rabbit monoclonal) Abcam RRID:AB_2904231 UDD2 (1:1000) Antibody Anti- Rab10 (mouse monoclonal) Nanotools RRID:AB_2921226 0680- 100/Rab10605B11 (1:1000) Antibody Anti- Rab10 (phospho T73) (rabbit monoclonal) Abcam RRID:AB_2811274 ab230261 (1:1000) Antibody Anti- FLAG M2 (mouse monoclonal) MilliporeSigma RRID:AB_262044 F- 1804 (1:2000) Strain, strain background (Escherichia coli) E. coli DH5α Thermo Fisher 18258012 Strain, strain background (E. coli) E. coli STBL3 Thermo Fisher C737303 Strain, strain background (E. coli) E. coli Rosetta DE3 pLys Millipore 70956 Cell line (Homo sapiens) HeLa ATCC CCL- 2 Cell line (H. sapiens) HEK293T ATCC CRL- 3216 Chemical compound, drug MLi- 2 MRC PPU Chemical compound, drug Creatine phosphate Fluka Analytical #27920 20 mM Commercial assay or kit RED- NHS 2nd Generation (Amine Reactive) Protein Labeling Kit NanoTemper Technologies MO- L011 Commercial assay or kit CF 633 Succinimidyl Ester Protein Labeling Kit Biotium #92217 Other Creatine Phosphokinase Sigma C3755 30U Chemical compound, drug 18:1 (Δ9- Cis) PC (DOPC) Avanti Polar Lipids #850375 11 μmol Chemical compound, drug 18:1 PS (DOPS) Avanti Polar Lipids #840035 5 μmol Chemical compound, drug 18:1 DGS- NTA(Ni) Avanti Polar Lipids #790404 0.85 μmol Chemical compound, drug 18:1 PI(4)P Avanti Polar Lipids #850151 0.15 μmol Chemical compound, drug DiD Thermo Fisher D7757 0.01 μmol Recombinant DNA reagent pNIC Bsa- 4 His- Sumo Rab10 Q68L 1–181 Gift of Amir Khan Human Recombinant DNA reagent pET15b His- Mst3 Gift of Amir Khan Human Recombinant DNA reagent pET21b GFP- Rab10 Q68L- His Addgene RRID:Addgene_186015 186015 Human Recombinant DNA reagent pET21b His Rab8A Q67L Addgene RRID:Addgene_186014 186014 Human Vides et al. eLife 2022;11:e79771.

Techniques: Phospho-proteomics, Western Blot, Inhibition