Journal: STAR Protocols

Article Title: Using piggyBac transposon gene expression vectors to transfect Zscan5b gene into mouse pluripotent stem cells

doi: 10.1016/j.xpro.2021.100811

Figure Lengend Snippet: Transfecting transposon-expressing vector with mouse induced pluripotent stem cells (miPSCs) by electroporation (A) miPSCs are dissociated into pieces by trypsin. (B) Pellets are mixed with transposase-expressing vector and the piggyBac transposon gene expression vector (pPB-CAG-mZscan5b-P2A-EGFP) ( Niwa et al., 1991 ) in 100 μL Opti-MEM. (C–E) The vectors are transfected to miPSCs by electroporation using NEPA21 (NepaGene) under the following conditions: Poring pulse: 125 V, 5ms-pulse width, 50-ms pulse interval, two pulses, 10% attenuation rate, +; Transfer pulse: 20 V, 50-ms pulse width, 50-ms pulse interval, five pulses, 40% attenuation rate, +/−. The piggyBac transposon gene expression vector in t2i/L medium is then moved into the cytoplasm with a few transfer pulses with a low voltage and long duration after the poring pulse. (F) The electroporated cells are plated onto a 6-well MEF plate in mouse t2i/L medium.

Article Snippet: piggyBac transposon gene expression vector (pPB-CAG-mZscan5b-T2A-EGFP) , VectorBuilder , VB181015-1188bhq.

Techniques: Expressing, Plasmid Preparation, Electroporation, Transfection

Journal: STAR Protocols

Article Title: Using piggyBac transposon gene expression vectors to transfect Zscan5b gene into mouse pluripotent stem cells

doi: 10.1016/j.xpro.2021.100811

Figure Lengend Snippet: Green fluorescent protein (GFP)-positive colony picking After transfecting transposon-expressing vector with mouse induced pluripotent stem cells (miPSCs) by electroporation, around 20% of colonies are GFP-positive (A and B). After colony picking and fluorescence-activated cell sorting (FACS), the ratio of GFP-positive colony gradually increases (C and D).

Article Snippet: piggyBac transposon gene expression vector (pPB-CAG-mZscan5b-T2A-EGFP) , VectorBuilder , VB181015-1188bhq.

Techniques: Expressing, Plasmid Preparation, Electroporation, Fluorescence, FACS

Journal: STAR Protocols

Article Title: Using piggyBac transposon gene expression vectors to transfect Zscan5b gene into mouse pluripotent stem cells

doi: 10.1016/j.xpro.2021.100811

Figure Lengend Snippet:

Article Snippet: piggyBac transposon gene expression vector (pPB-CAG-mZscan5b-T2A-EGFP) , VectorBuilder , VB181015-1188bhq.

Techniques: Recombinant, Expressing, Plasmid Preparation, Staining, Electroporation

Journal: bioRxiv

Article Title: A STING-CASM-GABARAP Pathway Activates LRRK2 at Lysosomes

doi: 10.1101/2023.10.31.564602

Figure Lengend Snippet: (A) Schematic diagram summarizing the activators and inhibitors of the CASM pathway that were used in experiments in this figure. (B) Immunoblots illustrating the impact of DMXAA treatment on LC3B and GABARAP lipidation in RAW264.7 cells. i = unlipidated, ii = lipidated. (C) Immunoblots displaying the effects of a 2-hour treatment with DMSO versus DMXAA on the indicated proteins in wildtype and ATG16L1 knockout cells. (D) Immunoblot results comparing control versus saliphenylhalimide (Salip) treated cells following a 2-hour treatment. (E) Quantification of the phospho-Rab10/total Rab10 ratio from panel D. A Two-Tailed Unpaired Welch’s t test was performed. P = 0.0175. (F) Immunoblots depicting the impact of folimycin (added 1 hour before DMXAA and maintained throughout DMXAA incubation) on the response to a 2-hour treatment with DMSO versus DMXAA on the indicated proteins. (G) Immunoblots from wildtype RAW 264.7 cells versus cells that stably express mCherry-SopF after a 2-hour treatment with DMSO alone versus DMXAA, showing the impact on the indicated proteins in RAW 264.7 cells. (H) Quantification of the phospho-Rab/Rab10 ratios from panel G. A One-Way ANOVA with Sidak’s post-test was performed. For WT DMSO vs WT DMXAA, P = 0.0064. For WT DMXAA vs SopF DMXAA, P = 0.0011. Molecular weight markers (kDa) are indicated on the right side of each blot. Error bars represent standard deviations. All experiments were conducted in RAW 264.7 cells, and the data presented in this figure is representative of results obtained from a minimum of three independent experiments.

Article Snippet: Custom murine STING, human HALO-LRRK2, and human HA-GABARAP piggybac vectors were purchased from VectorBuilder.

Techniques: Western Blot, Knock-Out, Two Tailed Test, Incubation, Stable Transfection, Molecular Weight

Journal: bioRxiv

Article Title: A STING-CASM-GABARAP Pathway Activates LRRK2 at Lysosomes

doi: 10.1101/2023.10.31.564602

Figure Lengend Snippet: (A) Immunoblot results showing the impact of the indicated siRNAs on Rab10 phosphorylation and additional specified proteins in response to DMXAA. Experiments were performed 48 hours after siRNA transfections. (B) Immunoblots demonstrating the effects of a 2-hour treatment with DMXAA on the indicated proteins in wildtype and GABARAP knockout cells. (C) Quantification of the phospho-Rab10/Rab10 ratio in panel B. A One-Way ANOVA with Sidak’s post-test was performed. For WT DMSO vs WT DMXAA, P = 0.0064. For WT DMXAA vs GABARAP KO DMXAA, P = 0.0011. (D) Immunoblots demonstrating the influence of GABARAP knockout on the phospho-Rab10/Rab10 ratio in response to ML SA1 (2-hour treatment). (E) Immunoblots revealing the effect of GABARAP knockout on the phospho-Rab10/Rab10 ratio in response to Nigericin (2-hour treatment). (F) Immunoblots of whole cell lysates (WCL) and SPION-purified lysosomes from wildtype and GABARAP knockout cells treated with DMSO or DMXAA for 2 hours. (G) Quantification of lysosomal LRRK2 abundance (LRRK2 normalized to LAMP1) from panel F. A One-Way ANOVA with Sidak’s post-test was performed. For WT DMSO vs WT DMXAA, P = 0.0002. For WT DMXAA vs GABARAP KO DMXAA, P = 0.0002. (H) Immunoblots of the starting material (Inputs) and anti-HA immunoprecipitates from LRRK2 KO cells that stably express HALO-tagged human LRRK2 +/- HA-tagged human GABARAP, treated for 2 hours with vehicle (DMSO) or DMXAA. (I) Quantification of HALO-LRRK2 abundance in HA-GABARAP immunoprecipitations +/- STING activation with DMXAA. A Two-Tailed Unpaired Welch’s t test was performed. P = 0.0243. Molecular weight markers (kDa) are indicated on the right side of each blot. Error bars represent standard deviations. All experiments were conducted in RAW 264.7 cells, and the data presented in this figure is representative of results obtained from a minimum of three independent experiments, except for panel A where n=2.

Article Snippet: Custom murine STING, human HALO-LRRK2, and human HA-GABARAP piggybac vectors were purchased from VectorBuilder.

Techniques: Western Blot, Transfection, Knock-Out, Purification, Stable Transfection, Activation Assay, Two Tailed Test, Molecular Weight