gfp dynamin2 k44a  (Addgene inc)

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Western Blot, Phospho-proteomics, Positive Control, Inhibition, Control, Comparison, Live Cell Imaging, Expressing, Staining, Mutagenesis

Journal: Stem Cell Reports

Article Title: Clathrin-Mediated Endocytosis Regulates a Balance between Opposing Signals to Maintain the Pluripotent State of Embryonic Stem Cells

doi: 10.1016/j.stemcr.2018.11.018

Figure Lengend Snippet: CME Is Required for the Maintenance of mESC Pluripotency and Self-Renewal (A) Dot plot showing the results of the endocytic siRNA screen in mESCs, based on AP staining. Knockdowns resulting in a significant decrease in AP staining are marked in blue, while those resulting in an increase in AP staining are shown in red. (B) Bright-field images show AP staining and morphology of mESCs 2 days post Cltc knockdown, or expression of K44A Dnm ( K44A ). Scale bar, 50 μm. NTi, non-targeting siRNA control; Cltc i, Cltc siRNA; control, vector control. (C) Bar graph showing mESC colony number, 3 days post indicated conditions. (D and E) Line graph showing the proliferation rate of (D) mESCs and (E) MEFs over 3 days post indicated conditions. (F) Cell-cycle analysis of mESCs and MEFs 3 days post indicated conditions. Bar graph shows the percent of cells in G1, S, and G2 phases of the cell cycle for both mESCs and MEFs. (G) Bar graph showing the expression of pluripotency markers in mESCs under indicated conditions relative to control (n = 3). Control is shown as a dotted line at 1. (H) Bar graph showing the expression of differentiation markers in embryoid bodies generated from Cltc knockdown mESCs by qRT-PCR analysis (n = 3). SCi, scrambled shRNA control; Cltc i, Cltc shRNA. Error bars represent mean ± SD from three independent experiments (n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 by two-tailed Student’s t test.

Article Snippet: K44A Dynamin2-GFP mutant was obtained from Addgene (no. 34687).

Techniques: Staining, Knockdown, Expressing, Control, Plasmid Preparation, Cell Cycle Assay, Generated, Quantitative RT-PCR, shRNA, Two Tailed Test