Following gene ontology analysis of protoarray hits, proteins categorized as membrane bound organelles were identified. Potential hits in addition to CSNK1A1, ARHGEF7 and NTC were used to order a custom pooled ON-TARGETplus siRNA library (Dharmacon). HEK293FT cells were plated in 96 well plates and reverse transfected with siRNA at a final concentration of 50nM. Twenty-four hours later, cells were transfected with 3xFlag WT LRRK2 together with either 2xmyc WT RAB29 or 2xmyc Q67L RAB29 using Lipofectamine 2000. Thirty hours following plasmid transfection, cells were fixed and stained for M2-Flag, Myc (clone 9E1) and TGN46 before labeling with fluorescent secondary antibodies and Hoechst 33342 (Thermo Scientific). Cells were imaged at 20x objective using a Cellomics VTI Arrayscanner and the percentage of cells with Flag WT LRRK2 and TGN46 positive spots were recorded. For each siRNA, 6 wells containing a minimum of 1000 cells each were analyzed. Samples were compared to NTC siRNA control and ranked.
On targetplus sirna library
Manufactured by Horizon Discovery
Sourced in United States
The ON-TARGETplus siRNA library is a collection of small interfering RNA (siRNA) molecules designed to target and silence specific genes. Each siRNA in the library is optimized for potency and specificity to ensure efficient knockdown of its target. The library provides a comprehensive tool for investigating gene function and exploring potential therapeutic targets.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Vti arrayscanner, by Thermo Fisher Scientific (2 mentions)
Multidrop combi, by Thermo Fisher Scientific (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Ensight, by PerkinElmer (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Cetuximab, by Merck Group (1 mentions)
Capivasertib, by Selleck Chemicals (1 mentions)
Compound library, by Selleck Chemicals (1 mentions)
Abt 737, by Selleck Chemicals (1 mentions)
Abt 263, by Adooq (1 mentions)
Crizotinib, by Pfizer (1 mentions)
Acy 1215, by Selleck Chemicals (1 mentions)
Trametinib, by Selleck Chemicals (1 mentions)
Carfilzomib, by Selleck Chemicals (1 mentions)
Sirnas, by Qiagen (1 mentions)
Azd6244, by AstraZeneca (1 mentions)
8 protocols using on targetplus sirna library
1
LRRK2 Regulation of Rab29 Localization
2
Screening E3 Ligase siRNA Library in HEK293T Cells
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The ON-TARGETplus siRNA library containing 386 E3 ligase siRNA pools was purchased from Dharmacon (GE Healthcare, catalog G-105635- 01; Human ON-TARGETplus siRNA library-Ubiquitin Conjugation Subset 3-SMARTpool). HEK293T cells were plated in 96-well plates (4 × 103 cells/well). The transfection reagent (Lipofectamine 3000 Transfection Reagent) was diluted in Opti-MEM and transferred to each well of the siRNA pool. After 15 minutes of incubation, the siRNA (50 nM)/Opti-MEM/Lipo3000 mixture was transferred to the HEK293T cells. At 48 hours after transfection, the cells were harvested and the lysates were used for immunoblotting analysis of endogenous FOXM1 expression. The band intensities were measured by ImageJ (NIH). The siRNA sequences for E3 ligases are provided in Supplemental Table 1 .
3
siRNA Screening for Cell Viability
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siRNA-based screening was performed for the selected 55 genes in HCT116 cells. The siRNAs were selected from Dharmacon Human ON-TARGET plus siRNA Library. To eliminate the off-target effects, four independent siRNAs (SMARTpools) were used for each gene. siNC and siTOX were selected as the negative and positive controls in the screening assay. 0.15 μL lipofectamine RNAiMAX reagent (Thermo) was added to the siRNA-loaded 96-well plates using Multidrop Combi (Thermo). Then, HCT116 cells were seeded in the 96-well plates at a density of 1500 cells/well. The number of cells was counted in the following 4 days using Ensight (Perkin Elmer). Subsequently, CTG (G756A, Promega) was added and measured on an Envision plate reader (Perkin Elmer).
4
Comprehensive Research Protocol Inventory
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AZ’1569 (compound-43), AZ’8037 (compound-25; ref. 11 (link)), AZD1480, and AZD6244 (Selumetinib/ARRY-142866) were obtained from AstraZeneca, 5-fluorouracil (5-FU) and oxaliplatin from the Belfast City Hospital Pharmacy, cetuximab from Merck Serono, and crizotinib from Pfizer. Ruloxitinib, capivasertib, ABT-737, and compound library were purchased from Selleckchem, S6K-18 and ABT-263 from Adooq Biosciences, and SN-38 from Abatra. See Supplementary Materials and Methods for references of non–FDA-approved drugs used. siRNA targeting BCL2L1 and the ON-TARGETplus siRNA library was obtained from Dharmacon. See Supplementary Materials and Methods for details of plasmids.
5
LRRK2 Regulation of Rab29 Localization
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Following gene ontology analysis of protoarray hits, proteins categorized as membrane bound organelles were identified. Potential hits in addition to CSNK1A1, ARHGEF7 and NTC were used to order a custom pooled ON-TARGETplus siRNA library (Dharmacon). HEK293FT cells were plated in 96 well plates and reverse transfected with siRNA at a final concentration of 50nM. Twenty-four hours later, cells were transfected with 3xFlag WT LRRK2 together with either 2xmyc WT RAB29 or 2xmyc Q67L RAB29 using Lipofectamine 2000. Thirty hours following plasmid transfection, cells were fixed and stained for M2-Flag, Myc (clone 9E1) and TGN46 before labeling with fluorescent secondary antibodies and Hoechst 33342 (Thermo Scientific). Cells were imaged at 20x objective using a Cellomics VTI Arrayscanner and the percentage of cells with Flag WT LRRK2 and TGN46 positive spots were recorded. For each siRNA, 6 wells containing a minimum of 1000 cells each were analyzed. Samples were compared to NTC siRNA control and ranked.
6
Identifying E3 Ubiquitin Ligases in STING Pathway
7
siRNA Screening for Cell Viability
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siRNA-based screening was performed for the selected 55 genes in HCT116 cells. The siRNAs were selected from Dharmacon Human ON-TARGET plus siRNA Library. To eliminate the off-target effects, four independent siRNAs (SMARTpools) were used for each gene. siNC and siTOX were selected as the negative and positive controls in the screening assay. 0.15 μL lipofectamine RNAiMAX reagent (Thermo) was added to the siRNA-loaded 96-well plates using Multidrop Combi (Thermo). Then, HCT116 cells were seeded in the 96-well plates at a density of 1500 cells/well. The number of cells was counted in the following 4 days using Ensight (Perkin Elmer). Subsequently, CTG (G756A, Promega) was added and measured on an Envision plate reader (Perkin Elmer).
8
Targeting of ER Stress in Cancer
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