Fig 1: Functional analysis of Blastocystis ST7 EV proteome. (A) Table comparing proteins present in ST7 EVs and whole cell lysate. Proteins were identified using mass spectrophotometry. Protein function predicted using gene ontology analysis via the PANTHER database. (B) Venn diagram showing overlap between ST7B/ST7H EV proteins identified in this study, Entamoeba histolytica EVs identified by Sharma et al., 2020, and the top 100 EV markers as curated by the Vesiclepedia database. (C) Western blot analysis of endosomal sorting (RAB5C; 23 KDa), tumour suppressor gene (TSG) 101 (TSG101; 45KDa) and enrichment of vesicle trafficking (PDCD6IP/ALIX; 97 KDa) markers in isolated Blastocystis ST7 EVs and lysates (L), with apolipoprotein ApoA1 (31 KDa) serving as negative marker control (Thery et al., 2018). Each lane was loaded with 200 µg EV protein. Western blot shown here is representative of three runs.
Fig 2: Generation and characterisation of an EmGFP-RAB5C HeLa line. A, Sequence encoding an N-terminal EmGFP tag was integrated into the endogenous RAB5C locus by CHoP-In. B, A sense strand gRNA and PAM site was selected immediately upstream of the RAB5C start codon and CHoP-In primers were designed to amplify DNA encoding EmGFP, flanked by the necessary gRNA and PAM sites for intracellular cleavage and NHEJ-mediated integration. C, Following transfection with px330-RAB5C together with PCR donor fragments consisting of a frame corrected EmGFP tag without any Cas9 recognition site (no recognition seq.) or a full CHoP-In donor fragment (ChoP-In), cells were analysed and sorted by flow cytometry. WT cells are untransfected HeLa. Data are shown as FACS plots from individual experiments as well as mean data (+/- SD) from three independent experiments. D, Fluorescence microscopy revealed EmGFP signal (green) in cells from this mixed population which colocalised well with Alexafluor-555 labelled endocytic tracer transferrin (red), following uptake of the marker for 15 minutes in order to label early endosomes (scale bar equals 10 µm). E, Immunoblotting with an antibody against RAB5C revealed the presence of a higher molecular weight band corresponding to the EmGFP-RAB5C fusion in lysates from the mixed population. F, Sanger sequencing of integration junctions showed the fidelity of NHEJ mediated knock-in of EmGFP into the RAB5C locus
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