gfp rab7 t22n  (Addgene inc)

Journal: Nature Communications

Article Title: Endosomal trafficking participates in lipid droplet catabolism to maintain lipid homeostasis

doi: 10.1038/s41467-025-57038-8

Figure Lengend Snippet: A The knockdown efficiency of Rab7a in A549 cells was assessed by western blot analysis ( p < 0.0001). B Control or Rab7a-knockdown A549 cells were fixed by 4% PFA, and then stained by Bodipy 493/503 (1 μM) and DAPI (1 μg/mL) for 30 min. The number and size of LDs, and fluorescence intensity of Bodipy 493/503 were quantified by ImageJ ( n = 30 for all figures; p < 0.0001 for number of LDs and relative Bodipy fluorescence, p = 0.3763 for average size of LDs). C Triglyceride level in control or Rab7a-knockdown A549 cells was determined ( p < 0.0001). D A549 cells were transiently transfected with GFP-Rab7a, GFP-Rab7a-T22N, or GFP-Rab7a-Q67L, and then stained with Nile red (1 μM) for 30 min. The number and size of LDs, and fluorescence intensity of Nile Red were quantified by ImageJ ( n = 13 for all figures; for number of LDs, p = 0.0097 for Rab7a T22N, p < 0.0001 for Rab7a Q67L; for average size of LDs, p = 0.4779 for Rab7a T22N, p < 0.0001 for Rab7a Q67L; and for relative Bodipy fluorescence, p < 0.0001 for both Rab7a T22N and Rab7a Q67L). Images were captured by a Zeiss 880 microscope with a 63x objective lens. The scale bar is 5 μm. The graphs represented data from three independent experiments. The statistical significance of differences was determined by using the unpaired two-tailed student’s t test, and data quantifications were expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001; ns: no significance.

Article Snippet: pEGFP-C1-ADRP (addgene, #87161), mcherry-ACSL3 (addgene, #87158), GFP-Rab5 (addgene, #174454), mcherry-Rab5 (addgene, #55126), mcherry-Rab5 S23N, GFP-Rab7 (addgene, #61803), mcherry-Rab7 (addgene, #55127), GFP-Rab7 T22N (addgene, #28048), GFP-Rab7 Q67L (addgene, #28049), pCDNA3.1-Twinstrep Rab5 (Elife.

Techniques: Knockdown, Western Blot, Control, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1038/s41467-024-55052-w

Figure Lengend Snippet: a Schematic representation of identified proteins within enriched GO terms. Color coding indicates the time point(s) at which a given protein is enriched (Green 20 min; Red 1 hour; Blue 4 hour). Dashed lines between proteins specify physical interactions analyzed with STRING; grey rectangle depicts proteins involved in both processes. All indicated proteins are statistically significant ( p value < 0.05). Asterisks indicate that the Log fold change enrichment at that time point is smaller than 1. b – e Confocal images of neurons co-labeled for RUSH-LAMP1-V5 and STX6 ( b ), RAB11 ( c ), RAB7 ( d ), and LAMTOR4 ( e ) after 1 h or 4 h of biotin addition. Magnified regions from soma are depicted with orange boxes. Orange arrows indicate the region used to generate intensity profile graphs. Scale bar, 10 µm, and magnified images, 1 µm. Representative images in b – e were repeated in at least 2−3 independent experiments. See also Supplementary Figs. and , and Source Data 1. Source data are provided as a Source Data file.

Article Snippet: The following vectors were used: FUGW was a gift from David Baltimore (Addgene plasmid # 14883) , pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453) , psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmids # 12260 and # 12259) pmScarlet-i_C1 was a gift from Dorus Gadella (Addgene plasmid # 85044) , H2B-mNeonGreen-IRESpuro2 was a gift from Daniel Gerlich (Addgene plasmid # 183745) , LAMP1-RFP was a gift from Walther Mothes (Addgene plasmid # 1817) , pEGFP-VAMP4 was a gift from Thierry Galli (Addgene plasmid # 42313) , Str-KDEL_SBP-EGFP-E-cadherin was a gift from Franck Perez (Addgene plasmid # 65286) , mito-V5-APEX2 was a gift from Alice Ting (Addgene plasmid # 72480) , pAAV hSyn GFP-FXR1 was a gift from Martin Beaulieu (Addgene plasmid # 112732) , LAMP1-GFP was a gift from Dr. Juan Bonifacino, GFP-RAB6A, GFP-RAB7A and GFP-RAB11A were gifts from Casper Hoogenraad , pAAV ORANGE Gria1-HaloTag was a gift from Harold MacGillavry, PB-Ef1a-PCP-Halo (Addgene plasmid # 198337) and PB-Ef1a-β-actin-UTR-PP7 mRNA were gifts from Michael Ward.

Techniques: Labeling