Figure 5

Validation of anti-AQP11 antibody and tissue immunoblotting. (A) – (C) Confocal images of cultured CHO cells transiently transfected with rat AQP11 (A, B) or with empty vector (C) and stained with anti-AQP11 C-term antibody. Bars represent 20 μm (A, C) and 5 μm (B). (D) CHO cells were transiently transfected with rat AQP11, empty vector, or rat AQP4 M23 and treated with membrane impermeable biotin, then precipitated with streptavidin. Surface proteins eluted from the streptavidin (S) and cytosolic proteins from the supernatant (C) were analyzed by SDS-PAGE followed by immunoblotting with antibodies against AQP11 and actin. As a control, cells transfected with AQP11 were precipitated with streptavidin but not incubated with biotin. (E) Western blot of multiple rat tissue probed with AQP11 antibody (upper panel). The bottom panel was probed with the same antibody preincubated with antigenic peptide. (F) Sucrose density sedimentation of AQP11 from rat brain. Membrane proteins from rat brain hemispheres were layered onto a 5–20% linear sucrose gradient, spun, and analyzed by SDS-PAGE followed by immunoblotting for either AQP4 or AQP11. Fraction 1 corresponds to the lightest fraction, 23 to the heaviest. Molecular size in kDa is shown.