arf1 mcherry  (Addgene inc)

Journal: Science Advances

Article Title: p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi

doi: 10.1126/sciadv.abf4885

Figure Lengend Snippet: ( A ) PAQR11-interacting proteins were identified by using a proximity-dependent biotin identification (BioID) assay. ( B ) A confocal micrograph demonstrates that a Golgi marker (GM130) colocalizes with a hemagglutinin (HA)–tagged BioID/PAQR11 fusion protein but not control vector (HA-BioID). ( C ) Venn diagram illustration of proteins identified by both methodologies (BioID assay on H1299 cells and N-terminal PAQR11 peptide pull-down assay on 344SQ cells). ( D ) Validation of hits identified by Western blot analysis of streptavidin beads isolated from H1299 cells transfected with HA-BioID or HA-BioID-PAQR11 and treated with (+) or without (−) 100 μM biotin. ( E ) Confocal micrographs of EGFP-tagged PAQR11 and mCherry-tagged ARF1 in 344SQ cells. Blue indicates DAPI. Scatter plot shows the percentage of mCherry-ARF1 that colocalizes with EGFP-PAQR11 in each cell (dot). Scale bar, 10 μm. ( F ) Confocal micrographs of ARF1-EGFP in PAQR11-deficient and -replete 344SQ cells. Red indicates Golgin-97, and blue indicates DAPI. Scale bars, 10 μm. Right: Scatter plot shows the percentage of Golgin-97 that colocalizes with ARF1 in each cell (dot). ( G ) Intensity–pseudo-colored confocal micrographs of Golgi-associated and cytosolic ARF1-EGFP. Scale bars, 10 μm. The scatter plot shows the Golgi/cytosolic ARF1-EGFP ratio in each cell (dots). Results represent means ± SD values from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test.

Article Snippet: The ARF1-mCherry expression vector was modified from ARF1-EGFP (Addgene plasmid #49578) by replacing the EGFP coding sequence with the mCherry sequence.

Techniques: Marker, Plasmid Preparation, Pull Down Assay, Western Blot, Isolation, Transfection, Two Tailed Test

Journal: Science Advances

Article Title: p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi

doi: 10.1126/sciadv.abf4885

Figure Lengend Snippet: ( A ) Western blot analysis of whole cell lysate (WCL) and vesicle- and Golgi-fractionated H1299 cell lysates to confirm enrichment of fractionated lysates in organelle-specific markers. ER, endoplasmic reticulum. ( B and C ) Western blot analysis of FAM3C and PLAU in WCL and Golgi- and vesicle-enriched fractions of siRNA-transfected H1299 cells (B). Protein levels relative to siCTL controls quantified densitometrically (C). ( D ) FAM3C and PLAU expression constructs. ( E ) Confocal micrographs of H1299 cells transfected with expression constructs. Vesicles containing ectopic FAM3C and PLAU were detected using fluorescent tags (EGFP-FAM3C) or antibodies against Flag (PLAU). Golgi localization determined on the basis of colocalization with the trans-Golgi marker Golgin-97. Nuclei were counterstained with DAPI (blue). Scale bar, 8 μm. ( F ) Contrast-adjusted confocal micrographs of H1299 cells cotransfected with Flag-PLAU and indicated siRNAs and stained with anti-Flag antibodies. Arrows indicate Flag-PLAU + vesicles. Dotted lines indicate cell boundary. Scale bar, 10 μm. Vesicle numbers per cell (dots) were quantified. ( G ) Schematic illustration of a working model in which p53 loss leads to PAQR11 up-regulation, initiating an ARF1-dependent secretory process that is amplified through an autocrine PLAUR-dependent signaling pathway. Results represent means ± SD values from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Article Snippet: The ARF1-mCherry expression vector was modified from ARF1-EGFP (Addgene plasmid #49578) by replacing the EGFP coding sequence with the mCherry sequence.

Techniques: Western Blot, Transfection, Expressing, Construct, Marker, Staining, Amplification, Two Tailed Test