Journal: The Journal of Biological Chemistry

Article Title: Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation *

doi: 10.1074/jbc.M114.622274

Figure Lengend Snippet: LH3 provides a cell surface docking mechanism for MMP-9 by recognizing its FN domain. A, mass spectrometry analysis. PLOD3_HUMAN (LH3) appeared to be specifically pulled down by MMP-9 and the FN domain according to mass spectrometry analysis with 95% probability (140% probability variance). Coomassie Blue staining of the pulldown of labeled MMP-9, FN, and ΔFN is shown (right panel). B, MMP-9 interacts with endogenous LH3 at the fibroblast cell surface via its FN domain. A representative anti-LH3 antibody immunoblot of anti-v5 antibody immunoprecipitates shows that endogenous LH3 is immunoprecipitated with both v5-tagged MMP-9 and its recombinant FN domain (v5 IP MMP-9 and FN), confirming the specificity of the interaction via fibronectin type II-like motifs. Anti-FLAG antibody immunoprecipitations (IP) constitute a control. C, interaction between MMP-9 and LH3 decreases upon LH3 depletion. PLA analysis between v5-tagged MMP-9 or ΔFN and endogenous LH3 in HSF showing specificity of the interaction between LH3 and the FN domain of MMP-9 (left panel) and impairment of the interaction when LH3 is depleted (KD) (right panel). nb, number. Results represent mean values ±S.E. (error bars). *, p ≤ 0.05.

Article Snippet: Chemical compounds used included:4-aminophenylmercuric acetate (164610, Calbiochem), Complete Mini EDTA-free protease inhibitors (11836170001, Roche Applied Science), FcR blocking reagent (130-059-901, Miltenyi Biotec), FuGENE 6 Transfection Reagent (E2692, Promega), Interferin (409-01, Polyplus Transfection), Sulfo-SBED Biotin Label Transfer Reagent (33034, Pierce), SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Scientific Pierce), human TGF-β1 (100-B-001, R&D Systems), Duolink II PLA Probe Anti-Mouse PLUS (DUO92001, Sigma-Aldrich), Duolink II PLA Probe Anti-Rabbit MINUS (DUO92005, Sigma-Aldrich), Duolink In Situ Detection Reagents Red (DUO92008, Sigma-Aldrich), and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) (human; three unique 27-mer siRNA duplexes) (SR305927, Origene).

Techniques: Mass Spectrometry, Staining, Labeling, Western Blot, Immunoprecipitation, Recombinant

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of VCP cofactors and their proposed functions

Article Snippet: FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR308083.

Techniques: Membrane

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: VCP cofactors differentially regulate tau seeding. VCP cofactors were either knocked out via CRISPR/Cas9 ( A-D ) or knocked down via siRNA ( E–G ) in v2L biosensors prior to exposure to increasing amounts of tau fibrils. A Knockout of FAF2 increased tau seeding whereas knockout of B ATXN3, C NSFL1C, and D UBE4B reduced tau seeding. P values: FAF2 (*** 0.0001, **** < 0.0001); ATXN3 (**** < 0.0001); NSFL1C (*** 0.0002); UBE4B (**** < 0.0001). E Knockdown of NGLY1, F NPLOC4, and G OTUB1, decreased tau seeding. P values: NGLY1(**** < 0.0001); NPLOC4 (**** < 0.0001); OTUB1 (*** 0.0004, **** < 0.0001, *** 0.0001). Graphs are representative of n = 3 independent experiments, with each data point derived from technical triplicate. Error bars represent S.D. Some error bars are too small to be visible. H Cofactor KO did not affect tau uptake. P values: ns = 0.9819, 0.9988, 0.9956, 0.9928, in order of bars on the graph. I Cofactor KD did not affect tau uptake. P values: ns = 0.9795, 0.1856, 0.3928, in order of bars on the graph. Error bars represent S.E.M ( n = 3). One-Way ANOVA with a 95% confidence interval

Article Snippet: FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR308083.

Techniques: CRISPR, Knock-Out, Knockdown, Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of Reagents

Article Snippet: FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR308083.

Techniques: Protease Inhibitor, Cell Culture, Transfection, Magnetic Beads, Sequencing, Modification

Journal: The Journal of Neuroscience

Article Title: Mutant Huntingtin Impairs BDNF Release from Astrocytes by Disrupting Conversion of Rab3a-GTP into Rab3a-GDP

doi: 10.1523/JNEUROSCI.0168-16.2016

Figure Lengend Snippet: mHtt associates with Rab3a in astrocytes. A, Knocking down Rab3a via siRNA in WT astrocytes. B, ELISA results showing that downregulation of Rab3a inhibits the release of BDNF and ATP from WT astrocytes compared with scrambled siRNA transfected astrocytes (Student's t test: BDNF release, n = 3 independent experiments, p = 0.0353; ATP release, n = 5 independent experiments, p = 0.0142). C, D, Association of mHtt with Rab3a is detected in cultured KI astrocytes (C) but not in cultured KI neurons (D). Endogenous mHtt in KI astrocytes or KI neurons was immunoprecipitated by 1C2 antibody, and the immunoprecipitates were probed with antibody to Rab3a. Immunoprecipitation with IgG served as a control. E, Association of mHtt with Rab3a-V5 was detected in cultured KI astrocytes infected with Rab3a-V5 adenovirus. Rab3a was immunoprecipitated by anti-V5 antibody, and the immunoprecipitates were probed with antibodies to 1C2 to detect mHtt. Immunoprecipitation with IgG served as a control. F, In vitro binding assay showed that in vitro translated Htt (1–212 aa) with 150Q binds to purified GST-Rab3a. G, Binding of mHtt to GTP-Rab3a was found in cultured HD KI astrocytes. To immunoprecipitate endogenous mHtt in HD KI astrocytes, 1C2 antibody was used, and the immunoprecipitates were probed with the antibody specific to GTP-Rab3a. *p < 0.05.

Article Snippet: Rab3a siRNA duplexes and negative control siRNA were purchased from OriGene (SR402766).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Cell Culture, Immunoprecipitation, Infection, In Vitro, Binding Assay, Purification

Journal: American Journal of Physiology - Cell Physiology

Article Title: (Pro)renin receptor mediates albumin-induced cellular responses: role of site-1 protease-derived soluble (pro)renin receptor in renal epithelial cells

doi: 10.1152/ajpcell.00006.2017

Figure Lengend Snippet: Sequences of custom-made siRNAs directed toward human targets

Article Snippet: The knockdown of endogenous furin (catalog no. SR303336), ADAM19 (catalog no. SR305748), proprotein convertase (PC) subtilisin/kexin type 7 (PCSK7; catalog no. SR306062), S1P (catalog no. SR305740), PCSK9 (catalog no. SR316837), or PRR (catalog no. SR306857) was performed using the predesigned small interfering RNA (siRNA) from OriGene (Rockville, MD).

Techniques: Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: (Pro)renin receptor mediates albumin-induced cellular responses: role of site-1 protease-derived soluble (pro)renin receptor in renal epithelial cells

doi: 10.1152/ajpcell.00006.2017

Figure Lengend Snippet: Primer sequences for real-time PCR

Article Snippet: The knockdown of endogenous furin (catalog no. SR303336), ADAM19 (catalog no. SR305748), proprotein convertase (PC) subtilisin/kexin type 7 (PCSK7; catalog no. SR306062), S1P (catalog no. SR305740), PCSK9 (catalog no. SR316837), or PRR (catalog no. SR306857) was performed using the predesigned small interfering RNA (siRNA) from OriGene (Rockville, MD).

Techniques: Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: (Pro)renin receptor mediates albumin-induced cellular responses: role of site-1 protease-derived soluble (pro)renin receptor in renal epithelial cells

doi: 10.1152/ajpcell.00006.2017

Figure Lengend Snippet: Effects of siRNA-mediated silencing of PCSK7, PCSK9, and S1P on PRR cleavage induced by BSA in HK-2 cells. The cells were transfected with siRNA against PCSK7, PCSK9, or S1P for 48 h and then treated with 20 mg/ml BSA for 24 h. A and B: immunoblotting of fPRR and sPRR. Shown are representative blots and quantitative densitometry analysis. C and D: ELISA detection of medium sPRR. The values are normalized by protein content. *P < 0.05 vs. CTR; #P < 0.05 vs. BSA; NS, nonsignificance vs. BSA. Data are means ± SD; n = 6/group. C and D values are presented as the median (central line), interquartile range (box), and range (whiskers).

Article Snippet: The knockdown of endogenous furin (catalog no. SR303336), ADAM19 (catalog no. SR305748), proprotein convertase (PC) subtilisin/kexin type 7 (PCSK7; catalog no. SR306062), S1P (catalog no. SR305740), PCSK9 (catalog no. SR316837), or PRR (catalog no. SR306857) was performed using the predesigned small interfering RNA (siRNA) from OriGene (Rockville, MD).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Life Science Alliance

Article Title: CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane

doi: 10.26508/lsa.202302045

Figure Lengend Snippet: (A) Diagram of RAB5, RAB11 siRNA, and Brefeldin A effect on CFTR trafficking. (B) RAB5 and RAB11 siRNA treatments. Lytic and extracellular assays were performed after 48 h of siRNA treatments. (C) Brefeldin A inhibition of protein transport from ER to GA. Lytic and extracellular assays were performed after 6 h treatment. The effect of siRNA and Brefeldin A treatments on viability was measured by MTS assay (mean ± SD). The luminescent signal was measured after 30 min (mean ± SD). All experiments were done in three independent biological replicates (n = 9; technical replicates). P -values were calculated using one-way ANOVA-Tukey’s test, ns = nonsignificant, * P ≤ 0.05, ** P < 0.005, *** P < 0.001.

Article Snippet: Antibodies: mouse anti-human CFTR antibodies (569, TJA9; CF Foundation), donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor 647; Invitrogen). siRNA: CFTR Human siRNA Oligo Duplex (Locus ID 1080; Origene), RAB5 (RAB5A) Human siRNA Oligo Duplex (Locus ID 5868), and RAB11 Human siRNA Oligo Duplex (Locus ID 8766).

Techniques: Inhibition, MTS Assay

Journal: Science (New York, N.Y.)

Article Title: A protein network map of head and neck cancer reveals PIK3CA mutant drug sensitivity

doi: 10.1126/science.abf2911

Figure Lengend Snippet: Key Resources

Article Snippet: Gαi-DREADD (pcDNA3.1) Antibodies RSK1/2/3 antibody Cell Signaling Technology 9355 ERK1/2 Cell Signaling Technology 4695 phospho-PAK1(S199/204)/PAK2(S192/197) Cell Signaling Technology 2605 PAK1 Cell Signaling Technology 2602 PAK2 Cell Signaling Technology 2608 pERK Cell Signaling Technology 9106 FGFR3 OriGene TA801078 Daple Millipore EMD ABS515 GAPDH Cell Signaling Technology 2118 secondary goat anti-rabbit HRP Southern Biotech 4010-05 P-HER3-Y1197 Cell Signaling Technology 4561 HER3 Cell Signaling Technology 12708 goat anti-mouse HRP Southern Biotech 1010-05 anti-B-tubulin Abcam ab6276 ERK Cell Signaling Technology 9102 Deposited data Unprocessed peptide files This paper PRIDE ProteomeXchange: PXD019469 Raw data This paper PRIDE ProteomeXchange: PXD019469 Chemicals, Peptides, and Recombinant Proteins Tris G-Biosciences RC108 Acetonitrile, HPLC grade (ACN) Thermo Fisher Scientific A955-4 cOmplete protease inhibitor cocktail tablets mini, EDTA-free Roche 11846 170 001 Dithiothreitol (DTT) Sigma-Aldrich 43819 Formic acid (FA) Thermo Fisher Scientific 28905 Iodoacetamide (IAA) Acros Organic 122270250 Sequencing-grade modified trypsin Promega V5111 Benzonase Sigma E1014-25KU Trifluoroacetic acid (TFA) Thermo Fisher Scientific 28904 Urea Sigma-Aldrich U5378-1kg Fetal bovine serum (FBS) Gibco A3160502 DMEM Corning MT10013CV DMEM/F12 Corning MT10092CV Water, HPLC grade Sigma-Aldrich 270733-4 L Igepal (NP-40) Sigma-Aldrich I3021 Minimal Essential Media Corning 10-009-CV Opti-MEM Thermo Fisher Scientific 31985062 BEGM™ (Lonza) Lonza CC-3170 1% Penicillin-Streptomycin Corning MT30002Cl Paraformaldehyde, 4% solution in PBS Thermo Scientific MFCD00133991 PolyJet SignaGen SL100688 Lipofectamine 3000 ThermoFisher Scientific L3000008 hydrocortisone Sigma H6909-10ML Rapigest Waters 186001861 3x Flag Peptide Sigma F4799-4MG Anti-Flag M2 Magnetic Beads Sigma M8823-5ML Lipofectamine RNAiMAX Thermo Fisher Scientific 100014472 siFGFR3 Sigma Aldrich SIHK0780, SIHK0781, SIHK0782 native coelenterazine Biotium 10110-1 pooled siControl Dharmacon D-001810-10-20 siDaple Dharmacon L-033364-01-0005 10μM clozapine-N-oxide Cayman Chemical NC1044836 5μM native coelenterazine Biotium 10110-1 RPS6KA1 siRNA pool OriGene SR304161 non-targeting control siRNA Dharmacon D-001810-10 Triton X-100 Thermo Scientific 9002-93-1 Software and Algorithms artMS Bioconductor https://www.bioconductor.org/packages/release/bioc/html/artMS.html MSstats Bioconductor https://bioconductor.org/packages/release/bioc/html/MSstats.html Skyline MacCoss Lab https://skyline.ms/project/home/begin.view?

Techniques: Mutagenesis, Recombinant, Protease Inhibitor, Sequencing, Modification, Magnetic Beads, Software, Mass Spectrometry