Fig 1: Blockage of autophagosome clearance and maturation of endosome and lysosomal proteases in the Vps18 CKO brains. A–D, Western blot analyses of proteins or markers involved in autophagy, endocytosis, and biosynthetic pathways using the cell lysates prepared from the whole brain of the Vps18 CKO and Ctrl mouse at P10. A and B, the accumulation of both forms of LC3 (LC3-I and LC3-II) (A) and ubiquitinated proteins (B). C, an increased expression of EEA1 and Rab7, but not Rab4 and Rab11. D, a dramatic accumulation of immature cathepsin D (52 and 48 kDa) and a marked reduction of the matured form (34 kDa). Total proteins were extracted from the Vps18 CKO or Ctrl mouse brains and blotted with the indicated antibodies. E, the accumulation of autophagosomes and lysosome/late endosomes in the Vps18 CKO brain cells. Cryosections of cerebral cortex from the indicated mice were immunofluorescently stained with anti-LC3 and anti-LAMP1. F, buildup of ubiquitin-positive inclusions in the Vps18 CKO brains at P10. Cryosections of various indicated brain tissues from Vps18 CKO and Ctrl mice were immunohistochemically stained with anti-ubiquitin antibody. Insets are enlarged views of the cropped regions. Arrows indicate ubiquitin-positive inclusions. G, co-IF staining with anti-ubiquitin and anti-p62 antibodies shows that ubiquitin-containing inclusion bodies in P10 Vps18-deficient brain cells are also positive for p62. Arrows indicate colocalized signals of ubiquitin and p62. Blots shown are representatives of three experiments with similar results. Images shown are representative mouse brain sections from at least three mice per group. (Scale bar: 20 μm (E); 50 μm (F); 5 μm (G).)
Fig 2: Rab4 expression in HC11 stem-like, pre-differentiated and differentiated cells. Rab4 expression analyzed by immunofluorescence in HC11 stem-like cells, pre-differentiated cells and differentiated cells.
Fig 3: MHCII localize predominantly to lysosomes, but not early and recycling endosomes. a–f Confocal micrographs of fixed double-fluorescent IFNγ-treated astrocytes displaying immunolabeled MHCII (red, left) and compartments labeled by the primary antibody against MHCII (a), LAMP1-EGFP (b), primary antibodies against Rab7 (c), Rab4A (d), EEA1 (e), TPC1 (f), and the corresponding fluorescent secondary antibodies (green, middle). The merged images display co-localized pixels (yellow, right); insets display a magnified view of the selected vesicles (white open frame). Scale bars: 20 μm (large images) and 1 μm (insets) (a–f). (g) Graph displaying quantitative co-localization (%, mean ± SEM) of anti-MHCII fluorescence versus anti-MHCII, LAMP1-EGFP, anti-Rab7, anti-Rab4A, anti-EEA1, and anti-TPC1 fluorescence. The numbers above the bars indicate the number of cell images analyzed
Fig 4: PKC inhibition induced redistribution of pPYK2 and AXL to endosomal/lysosomal compartment.The subcellular localization of pPYK2Y402 and AXL in control and PKC inhibitor (RO-31-8220)-treated (2 h) cells were assessed by IF analysis. Representative images of SUM159 cells are shown. Scale bar, 10 μm. Colocalization was estimated by the colocalization module of ZEN software (see the Materials and Methods section) and results are shown in Table S1. (A) Subcellular localization of AXL and pPYK2 with Lysotracker staining in control and RO-31-8220-treated SUM159 cells. (B, C) Subcellular localization of AXL (red) with the endosomal marker Rab4 (green) in control or RO-31-8220–treated SUM159 (B), or with the indicated endosomal/lysosomal markers (Rab11, CD9, and CD63) (green) in RO-31-8220–treated SUM159 (C). (D) Subcellular localization of pPYK2 (red) with CD9 (green) in RO-31-8220–treated SUM159. Scale bar, 10 μm.
Fig 5: α2M* induces LRP1 intracellular redistribution in MIO-M1 cells. The cellular distribution of LRP1, in untreated or α2M* treated (60 nM for 30 min) MIO-M1 cells, was compared to that of (a) Rab4-positive endosomes, (b) EEA1-positive early endosomes, (c) GFP-Rab7-positive late endosomes, and (d) LysoTracker-stained vesicles. Representative confocal micrographs (middle optical section planes) are shown, along with merge images showing the colocalization of LRP1 (red in a–c and green in d) with each specific intracellular marker (green or red as appropriate). The mask images depict colocalized pixels (white) in merge images and the mean of the Manders’ coefficients with their respective standard deviations (MC ± SD). Three independent experiments were performed and at least 50 cells were analyzed per condition. Asterisks (*) indicate significant differences (P < 0.05) relative to control.
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