Tuberin-Dependent Membrane Localization of Polycystin-1 Elena Kleymenova, Oxana Ibraghimov-Beskrovnaya, Hiroyuki Kugoh, Jeff Everitt, Hui Xu, Kaoru Kiguchi, Greg Landes, Peter Harris, Cheryl Walker  Molecular Cell  Volume 7, Issue 4, Pages 823-832 (April 2001) DOI: 10.1016/S1097-2765(01)00226-X

Figure 1 Affected Polycystic and Normal Kidneys from Eker Rats (A and B) Gross appearance of polycystic and normal kidneys, respectively, from 3-month-old Eker carrier (Tsc2Ek/+) males. Bars, 1 cm. (C and D) Sagittal sections of the kidney in panels A and B, respectively. Arrows indicate cysts Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)

Figure 2 Genetic Analysis of Tsc2-Pkd1 Locus in Affected Kidney Cells (A) PCR genotyping of affected and normal kidney cells. (B) Southern analysis of the Tsc2 gene in the EKT2 cell line. Lower panel shows equal DNA loading in both lines confirmed by reprobing of the blot with a probe for the rat IGF1 gene. (C and D) Southern analysis of the Pkd1 gene in the EKT2 cell line using 3′ XbaI and 5′ BglII Pkd1 polymorphisms, respectively. The 3′ probe recognizes 9.2 kb and 5.2 kb XbaI Pkd1 fragments adjacent to wild-type and mutant Tsc2 alleles, respectively. The 5′ probe detects 7.2 kb and 7.0 kb BglII fragments of Pkd1 alleles adjacent to wild-type and mutant Tsc2 alleles, respectively, and 8.1 kb fragment that is present in both Pkd1 alleles. The lower panels show equal DNA loading, as assessed by reprobing the blots with a probe for the rat IGF1 gene. (E) Northern analysis of normal rat kidneys and cell lines using the 5′ Pkd1 probe Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)

Figure 3 Western Analysis of Tuberin Expression (A) The 180 kDa tuberin-immunoreactive band was detected in the MDCK, TRKE2, and RKE but not in the ERC18, ERC15 and EKT2 cell lines. Nonspecific ∼80 kDa band was visible in all samples. (B) Tuberin expression in EKT2 cells after transient transfection with the sense Tsc2 expression construct (EKT2s) but not with the antisense Tsc2 expression construct (EKT2as). (C) Tuberin expression in ERC15 i cells (cotransfected with the lac repressor and the inducible Tsc2 constructs) after addition of 5 mM IPTG to the growth medium. Absence of tuberin in ERC15 lac cells (lac repressor construct alone) following addition of 5 mM IPTG. (D) Tuberin expression in TRKE2 cells after transfection with antisense Tsc2 expression construct. Equal intensity of a 80 kDa band indicates equal loading of samples Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)

Figure 4 Membrane Polycystin-1 Localization and Expression in Tuberin-Positive and Tuberin-Negative Cell Lines (A–C, and E) ICC with anti-LRR antibody of MDCK, EKT2, TRKE2, and ERC15 cells, respectively. Arrows indicate polycystin-1 localized to the lateral cell membrane in MDCK and TRKE2 cells. (D) Loss of membrane polycystin-1 staining in TRKE2 cells after incubation of anti-LRR antibody with a hundred-fold excess of LRR immunizing polypeptide. Bars, 10 μm. (F and G) ICC with anti-polycystin-1 antibody BD3 confirming membrane localization of polycystin in TRKE2 cells and absence of membrane-localized polycystin-1 in EKT2 cells. (H) Western analysis of polycystin-1 expression in crude membrane fractions. Analysis of polycystin-1 in RKE cells was performed using 300 μg of total cell lysate instead of the crude membrane fraction Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)

Figure 5 ICC of Cadherins in Tuberin-Negative EKT2 and ERC15 Cells (A) Membrane staining with a pan anti-cadherin antibody in tuberin-negative cystic EKT2 cells. (B) Computer-generated image of the lateral membranes following confocal analysis of EKT2 cells stained with a pan anti-cadherin antibody demonstrating the distribution of cadherins from the basal to apical aspect of the cells. (C) Lack of E-cadherin at the lateral cell membranes in EKT2 cells. (D) Positive staining for the lateral cell membrane-localized E-cadherin in tuberin-negative ERC15 cells. Bars, 10 μm Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)

Figure 6 Polycystin-1 Localization in the Renal Cell Lines Transfected with Sense and Antisense Tsc2 Expression Constructs (A and B) EKT2 cells transfected with sense and antisense (control) Tsc2 constructs, respectively. Arrow indicates polycystin-1 cell membrane localization. (C) ERC15 i cells cotransfected with lac repressor protein and inducible Tsc2 expression constructs grown in DF8 medium with 5 mM IPTG for 6 hr. Arrows indicate polycystin-1 cell membrane localization. (D) Same cells as in panel C after incubation of anti-LRR antibody with a hundred-fold excess of LRR immunizing peptide, which completely abolishes polycystin-1 membrane immunoreactivity. (E) ERC15 lac cells transfected with the lac repressor construct alone grown in DF8 medium with 5 mM IPTG for 6 hr have no detectable polycystin-1 membrane staining. (F) TRKE2 cells transfected with antisense Tsc2 construct exhibiting a significant reduction of polycystin-1 at the lateral membrane. (G) TRKE2 cells transfected with the sense Tsc2 construct (control) continue to exhibit lateral polycystin-1 staining. Bars, 10 μm Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)

Figure 7 Localization of Polycystin-1 within the Golgi and Effect of BFA Treatment on Polycystin-1 in Tuberin-Negative EKT2 Cells (A, B, and C) (A) Polycystin-1 (red) and (B) the p58 Golgi marker (green) co-localize (C, yellow) in EKT2 cells. Arrows indicate polycystin-1 and p58 localization. (D) EKT2 cells treated with 10 μg/ml BFA have disrupted Golgi as shown by Golgi marker p58 (red) and restoration of polycystin-1 localization to lateral cell membranes detected by anti-LRR antibody (green). Arrow indicates polycystin-1 membrane localization Molecular Cell 2001 7, 823-832DOI: (10.1016/S1097-2765(01)00226-X)