Supplemental Figures

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• Figure S1 - A: NaPi-IIc and PiT-2 protein levels are decreased in K-deficient rat kidney total cortical membranes. Rat kidney cortex homogenates were spun at 100,000 x g for 1 hour to recover all cellular membrane proteins. They were separated on SDS-PAGE and assayed by Western blotting for NaPi-IIa, NaPi-IIc, PiT-2 and ¦Â-actin. Samples from rats fed a K-deficient diet for 14 days (lanes 5-8) show increased NaPi-IIa but decreased NaPi-IIc and PiT-2 compared to samples from rats fed a control diet (lanes 1-4). The changes in NaPi-IIa, NaPi-IIc and PiT-2 in these total membrane preparations thus mimic the changes observed in the brush border membrane only (Figure 2). B: Isolated BBM do not contain early endosome membranes. BBM (lanes 1-8) or total membranes (lanes 9-16) containing equal amounts of total protein were separated by SDS-PAGE and probed for early endosomal antigen 1 (EEA1) and Rab5. No EEA1 was detected in the BBM, neither in control (lanes 1-4) nor in K-deficient (lanes 5-8) BBM. Some Rab5 was detected in the control BBM; however, this is not surprising since Rab5 has been shown to be associated with the apical plasma membrane in addition to early endocytic structures (Bucci C, Wandinger-Ness A, L¨¹tcke A, Chiariello M, Bruno Bruni C and Zerial M. Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells. Proc Natl Acad Sci U S A 91: 5061-5065, 1994). It is not clear why less Rab5 seems to be associated with the K-deficient BBM compared to the control BBM.
• Figure S2 - Apical region of an intercalated cell in a cortical collecting duct from a sample labeled for NaPi-IIc, demonstrating specificity of the immunolabel for the proximal tubule. No gold particles are present, which is representative of the findings in renal epithelial cells other than proximal tubule cells.