rabbit anti-cd71 (transferrin receptor) polyclonal antibody  (Assay Biotechnology)

Journal: American Journal of Physiology - Cell Physiology

Article Title: Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

doi: 10.1152/ajpcell.00118.2009

Figure Lengend Snippet: Colocalization of EGFP-NKCC1 vesicles with clathrin and the transferrin receptor (TfR) during PMA exposure. A: Western blot of whole MDCK cell lysate probed for clathrin using a rabbit anti-clathrin heavy chain (HC) antibody (1:200 dilution). Cells were cultured as described in Fig. 2 legend. B: colocalization of EGFP-NKCC1 with clathrin. Cells were cultured as described in Fig. 2 legend and exposed to 100 nM PMA for 0–10 min. Cells were double-labeled with rabbit Alexa Fluor 488-conjugated anti-GFP (green, 1:200 dilution) and rabbit anti-clathrin (1:200 dilution). Alexa Fluor 594-conjugated secondary antibody (red, 1:200 dilution) was used to visualize clathrin. Arrows show NKCC1 colocalization with clathrin in large vesicular structures at late time points and nonrecycling of clathrin to the membrane during PMA stimulation. Scale bar, 10 μm. C: percentage of NKCC1 vesicles colocalizing with clathrin staining calculated for each time point of B (n = 4 experiments, 90 cells). ***P < 0.001 vs. time 0 (unpaired t-test). D: colocalization of NKCC1 with the TfR. Cells were cultured as described in Fig. 2 legend and exposed to 100 nM PMA for 10 min. Cells were double-labeled with rabbit Alexa Fluor 488-conjugated anti-GFP (green, 1:200 dilution) and rabbit CD71 anti-TfR (1:200 dilution). Alexa Fluor 594-conjugated secondary antibody (red, 1:200 dilution) was used to visualize the TfR. Scale bar, 10 μm.

Article Snippet: Primary rabbit anti-caveolin-1 antibodies were obtained from Abcam (Cambridge, MA), rabbit anti-clathrin heavy chain antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), mouse anti-ZO-1 monoclonal antibody from Invitrogen (Carlsbad, CA), mouse anti-Na + -K + -ATPase α 1 -subunit from Millipore (Temecula, CA), rabbit polyclonal anti-CD71 anti-transferrin receptor (TfR) antibody from Novus Biologicals (Littleton, CO), and rabbit anti-dynamin II from Calbiochem.

Techniques: Western Blot, Cell Culture, Labeling, Membrane, Staining