Journal: Micromachines

Article Title: A Novel Electroporation System for Living Cell Staining and Membrane Dynamics Interrogation

doi: 10.3390/mi11080767

Figure Lengend Snippet: GFP-vinculin fluorescence images of a migrating NIH/3T3 fibroblast at different time points. The arrow indicates the moving (as well as the pseudopodia contraction) direction of the cell.

Article Snippet: GFP-mouse vinculin full length (889), a gift from Alpha Yap (Addgene plasmid # 67935; http://n2t.net/addgene:67935 ; RRID: Addgene_67935), was used for electrotransfection in this study.

Techniques: Fluorescence

Journal: Micromachines

Article Title: A Novel Electroporation System for Living Cell Staining and Membrane Dynamics Interrogation

doi: 10.3390/mi11080767

Figure Lengend Snippet: GFP-vinculin (green) and an F-actin fluorescence image of NIH/3T3 fibroblasts: a—cell with successful phalloidin-actin binding (orange); b—cell without phalloidin-actin binding; and c—an apoptotic cell (phalloidin appears in red without F-actin binding).

Article Snippet: GFP-mouse vinculin full length (889), a gift from Alpha Yap (Addgene plasmid # 67935; http://n2t.net/addgene:67935 ; RRID: Addgene_67935), was used for electrotransfection in this study.

Techniques: Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.

Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

Techniques: Membrane, Stable Transfection, Expressing, Ubiquitin Proteomics, Control, Western Blot, Quantitative Proteomics

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12 F36V -KRAS G12D , and the effector is MTH1-RNF152. B) Structure of LYMTAC-2 (FKBP12 F36V ligand-PEG2-MTH1 ligand). C) HiBiT cellular degradation assay in MTH1-RNF152 stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of three independent experiments. E) KRAS G12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.

Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

Techniques: Degradation Assay, Stable Transfection, Expressing, Knock-In, Western Blot, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of complex formation between KRAS G12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B) Structures of the pan-KRAS inhibitor and PROTAC C) Structures of LYMTAC-3 and LYMTAC-4. D) HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D ) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E) Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as INPUT. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Data are representative of three independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Data are representative of two independent experiments. H) HEK293T cells stably expressing HiBiT-FKBP12-KRAS G12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Data are representative of two independent experiments.

Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

Techniques: Degradation Assay, Stable Transfection, Expressing, Knock-In, Tube Formation Assay, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of Lysosome Membrane Proteins (LMPs). B) CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of three independent experiments. C) CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Data are representative of three independent experiments. D) HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of two independent experiments and reported as the mean ± S.E. E) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of two independent experiments. F) KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Data are representative of two independent experiments.

Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

Techniques: Membrane, Stable Transfection, Expressing, Control, Western Blot, Degradation Assay, Knock-In, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: Recurrent Loss of APOBEC3H Activity during Primate Evolution

doi: 10.1101/311878

Figure Lengend Snippet: A . Western blot analysis for the expression of AGM A3H haplotype 1, codon-optimized haplotype 1 A3H, rhesus macaque A3H, and codon-optimized rhesus macaque A3H. Vinculin was used as a protein loading control. Quantification was done relative to rhesus macaque A3H (normalized to 1). B . Top: Single-cycle infectivity assay of HIVΔvif in the presence of increasing amounts of A3H plasmid comparing codon-optimized AGM haplotype 1 A3H (black squares) and codon-optimized rhesus macaque A3H (open squares). Relative infection was normalized to viral infectivity in the absence of A3 proteins. Averages of three replicates, each with triplicate infections (± SEM) are shown. Bottom: Western blot analysis of protein expression level with amounts of plasmid added in panel B. β-Actin is shown as a loading control. C . Subcellular localization of rhesus macaque and AGM A3H haplotype 1 in HeLa cells. A3H proteins were detected with an anti-HA antibody (green) and DAPI staining was used to detect the nucleus (blue). Image is representative of n = 90 total images over 3 replicates.

Article Snippet: Lysates were quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, #23225) and 10 μg of protein was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-HA (BioLegend, #901503) and anti-actin (Sigma, #A2066) or anti-vinculin (Proteintech, #66305-1) antibodies at a 1:2000 dilution.

Techniques: Western Blot, Expressing, Infection, Plasmid Preparation, Staining