American Society of Hematology

Figure 5

$Figure 5. Rab7b is a small GTPase localized to late endosomes and lysosomes. (A) Distribution of Rab7b mutants in Raw 264.7 cells. Raw 264.7 cells were transiently transfected with His-tagged Rab7bT22N or Rab7b▵CC; 48 hours later, cells were immunostained with anti-His Ab and Orange Green 488-conjugated secondary Ab. (B) Examination of the subcellular localization of full-length Rab7b and Rab7b mutants in stable Raw 264.7 transfectants by Western blotting of the lysate, postnuclear supernatant, and membrane factions using anti-His Ab (upper panel). The lysate, postnuclear supernatant, and membrane fractions were also probed with indicated Abs (lower panel) to show the loading quantity and the fractionation specificity. (C-E) Confocal analysis of Rab7b subcellular localization. Raw 264.7 cells were transiently transfected with GFP-Rab7b plasmid; 48 hours later, cells were labeled with LysoTracker Red (D), or immunostained with first Ab against EEA-1 (C), TGN38 (C), or LAMP-1 (E) and Alexa Fluo 555-conjugated secondary Ab. The slides were finally examined under confocal microscopy, and representative images were presented. In panels D and E, the numerated regions were magnified and shown correspondingly at the right. (Bar = 100 μm).$

Rab7b is a small GTPase localized to late endosomes and lysosomes. (A) Distribution of Rab7b mutants in Raw 264.7 cells. Raw 264.7 cells were transiently transfected with His-tagged Rab7bT22N or Rab7b▵CC; 48 hours later, cells were immunostained with anti-His Ab and Orange Green 488-conjugated secondary Ab. (B) Examination of the subcellular localization of full-length Rab7b and Rab7b mutants in stable Raw 264.7 transfectants by Western blotting of the lysate, postnuclear supernatant, and membrane factions using anti-His Ab (upper panel). The lysate, postnuclear supernatant, and membrane fractions were also probed with indicated Abs (lower panel) to show the loading quantity and the fractionation specificity. (C-E) Confocal analysis of Rab7b subcellular localization. Raw 264.7 cells were transiently transfected with GFP-Rab7b plasmid; 48 hours later, cells were labeled with LysoTracker Red (D), or immunostained with first Ab against EEA-1 (C), TGN38 (C), or LAMP-1 (E) and Alexa Fluo 555-conjugated secondary Ab. The slides were finally examined under confocal microscopy, and representative images were presented. In panels D and E, the numerated regions were magnified and shown correspondingly at the right. (Bar = 100 μm).

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