mouse anti mklp1  (Santa Cruz Biotechnology)

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Immunofluorescence microscopy analysis of SW620 cells using anti-MKLP1 antibodies. SW620 cells cultured on glass coverslips were stained with annexin-V. Midbodies (MB) between prospective daughter cells do not stain with annexin-V ( top panel ). In contrast, midbody remnants (MB-R) associated with one of the cells stained with annexin V ( middle panel ). MB-Rs are also detected extracellularly as shed midbody remnants (sMB-Rs) ( bottom panel ). Inset panels represent enlarged images of sMB-Rs. Scale bar, 10 µm. b Immunofluorescence microscopy analysis of SW620-GAP-GFP cells using anti-MKLP1 antibodies. Nuclei (blue) were stained with Hoechst stain. Inset: higher magnification of GFP-tagged sMB-Rs in the extracellular space. Scale bar, 10 µm. c Immunofluorescence microscopy analysis of SW620, SW480 and SW1222 cells cultured in Matrigel TM matrix using anti-MKLP1 antibodies (green) and Alexa Fluor 594 Phalloidin (red) to stain actin. Nuclei (blue) were stained with Hoechst stain. White arrow heads point show sMB-Rs. Scale bar, 10 µm. d Immunohistochemistry analysis of normal human colon tissue and colon cancer tissue (adenocarcinoma) using anti-MKLP1 and anti-RACGAP1 antibodies. Red arrows indicate anti-MKLP1 or anti-RACGAP1 staining extracellular sMB-Rs. Images obtained from Human Protein Atlas ( http://www.proteinatlas.org/ ) with permission.

Article Snippet: Mouse antibodies against MKLP1 (Santa Cruz), RACGAP1 (Santa Cruz), ALIX (BD Biosciences), TSG101 (BD Biosciences), CD63 (Santa Cruz), CD81 (Santa Cruz), RAB2A (Thermo Fisher), FLOT1 (BD Biosciences), α-actinin (Abcam), CD9 (Santa Cruz) and HSP90 (BD Biosciences) were used.

Techniques: Immunofluorescence, Microscopy, Cell Culture, Staining, Immunohistochemistry

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Experimental workflow for purification of sMB-Rs from SW620 cell culture medium (CM). CM was subjected to differential centrifugation to obtain crude sMVs (10,000 × g pellet) and exosomes (100,000 × g pellet) that were further fractionated using isopycnic density gradient centrifugation. Photographic image shows that crude sMVs (10,000 × g pellet) floated in two major fractions: low-density fractions 7/8 (sMV-LD) and high-density fractions 9/10 (sMV-HD/sMB-Rs). b The buoyant densities of twelve 1-mL fractions collected for each preparation were determined by absorbance at 244 nm using a molar extinction coefficient of 320 L g −1 cm −1 . c SDS-PAGE of 12 OptiPrep™ fractions. Protein quantitation was determined by SYPRO Ruby staining and western blot analysis performed using indicated antibodies. d Western blot analysis of SW620 cell-derived Exos, 10,000 × g EVs (crude sMVs), sMV-LD (fractions 7-8) and sMB-R (sMV-HD) (fractions 9-10) using indicated antibodies ( n = 2 biological replicates). e Cryo-electron microscopic analysis of SW620 cell-derived Exos, sMV-LD and sMB-Rs. f Histogram represents the measurements of diameter of Exos, sMV-LD and sMB-R based on cryo-EM images. Data presented as mean ± s.e.m (standard error of mean). g Fluorescence microscopic analysis of Exos, sMVs-LD and sMB-Rs derived from SW620-GAP-GFP cells loaded onto aldehyde sulfate (AS) latex beads and immunostained with anti-MKLP1 antibodies (in red). h Bar plot showing protein yield (based on SYPRO Ruby protein quantitation) of Exos, sMVs-LD and sMB-R (sMVs-HD) secreted from five different cancer cell lines.

Article Snippet: Mouse antibodies against MKLP1 (Santa Cruz), RACGAP1 (Santa Cruz), ALIX (BD Biosciences), TSG101 (BD Biosciences), CD63 (Santa Cruz), CD81 (Santa Cruz), RAB2A (Thermo Fisher), FLOT1 (BD Biosciences), α-actinin (Abcam), CD9 (Santa Cruz) and HSP90 (BD Biosciences) were used.

Techniques: Purification, Cell Culture, Centrifugation, Gradient Centrifugation, SDS Page, Protein Quantitation, Staining, Western Blot, Derivative Assay, Cryo-EM Sample Prep, Fluorescence

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Venn diagram of proteins identified in SW620 cell-derived sMB-R (sMV-HD), sMV-LD and Exos. b Heatmap illustration of proteins identified in sMB-R (sMV-HD), sMV-LD and Exos. Proteins present in higher abundance in sMB-R (red) as compared to sMV-LD and Exos include conserved cytokinetic proteins as well as additional cytokinetic proteins. *Proteins uniquely identified in sMB-Rs. **Proteins enriched (fold change >2) in sMB-R compared to sMV-LD and Exos. c STRING-based protein-protein interaction network analysis of 928 enriched proteins in sMB-Rs (sMV-HD) compared to sMV-LD and Exos. The interactions were “evidence”-based, with “experiments” as active interaction source and interaction threshold set at 0.900 (highest confidence). Disconnected nodes in the network are hidden. Proteins identified under biological processes or molecular processes (Gene Ontology) are indicated. Centralspindlin complex components (RACGAP1 and KIF23/MKLP1) are also indicated. d EnrichmentMap of Reactome pathways enriched in 456 proteins commonly identified in SW620 cell-derived sMB-R proteome (2300 proteins) with the proteome of MB-Rs shed by Hela cells reported recently by Peterman et al. 2019 . e Mass spectrometry-based identification of KRAS peptides (UniProtKB ID RASK_HUMAN) in sMB-Rs. Two peptides (TEYKLVVVGAGGVGK and LVVVGAGGVGK) spanning Gly-12/ Val-12 substitution in KRAS protein. Peptide spectral profiles are displayed on the right. f Immunofluorescence microscopy of SW620 cells using anti-MKLP1 and anti-KRAS G12V antibodies. Nuclei (blue) were stained with Hoechst stain. White arrows indicate the position of MB and MB-Rs. Inset represents higher magnification. Scale bar, 10 µm. g Western blot analysis of exosomes, crude 10,000 x g sMVs, and isopycnic (iodixanol-density) gradient centrifugation fractions of sMV-LD and -HD/sMB-Rs using anti-KRAS G12V antibody.

Article Snippet: Mouse antibodies against MKLP1 (Santa Cruz), RACGAP1 (Santa Cruz), ALIX (BD Biosciences), TSG101 (BD Biosciences), CD63 (Santa Cruz), CD81 (Santa Cruz), RAB2A (Thermo Fisher), FLOT1 (BD Biosciences), α-actinin (Abcam), CD9 (Santa Cruz) and HSP90 (BD Biosciences) were used.

Techniques: Derivative Assay, Mass Spectrometry, Immunofluorescence, Microscopy, Staining, Western Blot, Gradient Centrifugation

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Uptake of sMB-Rs by fibroblasts. Fluorescence microscopy analysis of NIH3T3 fibroblasts incubated with/without SW620 cell-derived sMB-Rs or Exos (50 µg ml −1 ) for 2 h using anti-MKLP1 and anit-RACGAP1 antibodies. b Uptake and accumulation of sMB-Rs in NIH3T3 fibroblasts was quantified by counting MKLP1 + puncta per cell; data represented as mean ± s.e.m. Nuclei (blue) were stained with Hoechst. Scale bar, 10 µm. c Internalisation of sMB-Rs by fibroblasts. Confocal microscopy of NIH3T3 fibroblasts incubated with sMB-Rs using anti-MKLP1 (in green) and anti-RAB7 (in red) antibodies. Confocal microscopy analysis along Z-axis (inset) reveal internalisation of sMB-Rs following uptake. Scale bar, 10 µm. d Intercellular transfer of sMB-R KRAS G12V into NIH3T3 cells. Fluorescence microscopy of NIH3T3 fibroblasts incubated with SW620 cell-derived sMB-Rs (5 µg) for 2 h using anti-KRAS G12V antibodies. Nuclei were stained with Hoechst stain (blue). Right panel represents fluorescence signals from left panel overlaid onto bright-field images. Inset represents enlarged image. Scale bar, 10 µm.

Article Snippet: Mouse antibodies against MKLP1 (Santa Cruz), RACGAP1 (Santa Cruz), ALIX (BD Biosciences), TSG101 (BD Biosciences), CD63 (Santa Cruz), CD81 (Santa Cruz), RAB2A (Thermo Fisher), FLOT1 (BD Biosciences), α-actinin (Abcam), CD9 (Santa Cruz) and HSP90 (BD Biosciences) were used.

Techniques: Fluorescence, Microscopy, Incubation, Derivative Assay, Staining, Confocal Microscopy

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: Exosomes (class I EVs) are of endosomal origin (formed by invagination of multivesicular bodies (MVB)), shed microvesicles/ microparticles (class II extracellular vesicles) are formed via direct outward blebbing of plasma membrane and midbody remnants (MB-Rs) (class III extracellular vesicles) are generated by cytokinetic abscission of the interconnecting bridge between dividing daughter cells (post completion of cytokinesis). Current understanding of biophysical properties and stereotypic marker proteins for exosomes, sMVs (microparticles) and sMB-Rs are listed in the table. Centraspindlin complex proteins MKLP1 and RACGAP1 enable distinction of sMB-Rs from exosomes and sMVs.

Article Snippet: Mouse antibodies against MKLP1 (Santa Cruz), RACGAP1 (Santa Cruz), ALIX (BD Biosciences), TSG101 (BD Biosciences), CD63 (Santa Cruz), CD81 (Santa Cruz), RAB2A (Thermo Fisher), FLOT1 (BD Biosciences), α-actinin (Abcam), CD9 (Santa Cruz) and HSP90 (BD Biosciences) were used.

Techniques: Clinical Proteomics, Membrane, Generated, Marker