antibody against shank2  (Synaptic Systems)

Journal: Physiological Reports

Article Title: Shank2 Regulates Renal Albumin Endocytosis

doi: 10.14814/phy2.12510

Figure Lengend Snippet: Shank2 is expressed in podocytes in vivo and in vitro. (A) Immunofluorescence of Shank2 (green) in podocytes in rat glomerulus (arrows). Actin is stained blue. Bar – 10 μ m. (B) Immunofluorescence of Shank2 (green) in human podocytes. Actin is stained blue. We are unable to demonstrate Shank2 staining using IF in mouse podocytes because our Shank2 antibody does not work for IF on mouse tissue (but does work for western blot, see ). Bar – 10 μ m.

Article Snippet: Kidney sections were blocked (10% normal goat serum and 1% bovine serum albumin [BSA] in PBS) and incubated overnight at 4°C with primary antibody against Rb polyclonal Shank2 (1:100, Santa Cruz; Santa Cruz, CA).

Techniques: In Vivo, In Vitro, Immunofluorescence, Staining, Western Blot

Journal: Physiological Reports

Article Title: Shank2 Regulates Renal Albumin Endocytosis

doi: 10.14814/phy2.12510

Figure Lengend Snippet: Association of albumin-laden endosomes, distinct endosomal compartments, and Shank2. (A–C) Podocytes were treated with FITC-albumin ( green ; 10 min; 37°C), fixed and stained for Shank2 (r ed ) with (B) EEA1 ( blue ), (C) LAMP1 ( blue ), and F-Actin ( blue ). FITC-albumin partially colocalizes ( arrows ) with early endosomes and lysosomes. Shank2 also colocalizes ( arrows ) with FITC-albumin containing endosomes. Bar – 10 μ m, the same magnification was used for all images. (D–F) Intensity correlation coefficient analysis indicates there is a spatial dependency on the degree of colocalization of albumin with early endosomes and lysosomes, but not Shank2. Graph abscissas denote micron steps above the coverslip level. (D) Shank2 colocalization remained constant in all levels of the podocytes, but for (E) EEA1 and (F) LAMP1, colocalization diminished at the upper reaches of the podocytes.

Article Snippet: Kidney sections were blocked (10% normal goat serum and 1% bovine serum albumin [BSA] in PBS) and incubated overnight at 4°C with primary antibody against Rb polyclonal Shank2 (1:100, Santa Cruz; Santa Cruz, CA).

Techniques: Staining

Journal: Physiological Reports

Article Title: Shank2 Regulates Renal Albumin Endocytosis

doi: 10.14814/phy2.12510

Figure Lengend Snippet: Shank2 knockdown decreases albumin uptake in podocytes. (A) Western blot analysis of cultured human podocytes infected with nontarget control or Shank2 shRNA. Shank2 gives bands at ˜250 kD and 150 kD. (B) Densitometric analysis of Shank2 knockdown using lentiviral shRNA. Shank2 expression is significantly reduced to 46 ± 16% of control levels in podocytes infected with Shank2 shRNA ( P < 0.01) P value to figure. (C) Western blot analysis of FITC-albumin uptake in control (shControl) versus Shank2 knockdown (shShank2) podocytes. (D) Densitometric analysis of FITC-albumin uptake in control (shControl) versus Shank2 knockdown podocytes (shShank2). Intensities were normalized to shNTC 0 h condition. Shank2 knockdown decreased FITC-albumin uptake by 36 ± 23% compared to nontarget control ( P < 0.1) after 1 h of uptake and by 40 ± 24% compared to nontarget control after 2 h of uptake ( P < 0.07), although as noted these decreases did not reach statistical significance ( n = 3 experiments).

Article Snippet: Kidney sections were blocked (10% normal goat serum and 1% bovine serum albumin [BSA] in PBS) and incubated overnight at 4°C with primary antibody against Rb polyclonal Shank2 (1:100, Santa Cruz; Santa Cruz, CA).

Techniques: Knockdown, Western Blot, Cell Culture, Infection, Control, shRNA, Expressing

Journal: Physiological Reports

Article Title: Shank2 Regulates Renal Albumin Endocytosis

doi: 10.14814/phy2.12510

Figure Lengend Snippet: Shank2 knockout inhibits albumin uptake in podocytes. (A) Podocytes isolated from Shank2 knockout mice do not express Shank2. (B) Representative western blot of lysates from wild-type or Shank2 knockout podocytes incubated with FITC-albumin at 4°C (inhibits endocytosis, permits binding) or 37°C (permits endocytosis) for the times shown. (C) Densitometric analysis of the amount of FITC-albumin present in wild-type or Shank2 knockout podocyte cellular lysates. Intensities were normalized to the intensity at 37°C, 0 h for wild-type or Shank2 knockout podocytes. * P < 0.05, n = 3 independent experiments.

Article Snippet: Kidney sections were blocked (10% normal goat serum and 1% bovine serum albumin [BSA] in PBS) and incubated overnight at 4°C with primary antibody against Rb polyclonal Shank2 (1:100, Santa Cruz; Santa Cruz, CA).

Techniques: Knock-Out, Isolation, Western Blot, Incubation, Binding Assay

Journal: Physiological Reports

Article Title: Shank2 Regulates Renal Albumin Endocytosis

doi: 10.14814/phy2.12510

Figure Lengend Snippet: Albumin trafficking is impaired in Shank2 knockout mice in vivo. (A) Urinary albumin normalized to creatinine in wild-type ( n = 5) and Shank2 KO mice ( n = 7). (B) Shank2 knockout mice have no Shank2 expression in the renal cortex. (C) Transmission electron microscopy (TEM) demonstrates normal foot processes in wild-type mice (arrows). (D) In contrast, TEM shows broadening of podocyte foot processes in Shank2 KO and mild podocyte foot process effacement (arrowheads). (E) TEM demonstrates abundant endocytotic vesicles at the apical surface of a proximal tubule cell in a wild-type mouse (arrows). (F) There are significantly fewer vesicles (arrowheads) at the apical proximal tubular membrane of a Shank2 knockout mouse. (G) Intravital multiphoton images taken 15 min after FITC-albumin injection demonstrate FITC-albumin below the brush border in renal tubular cells in a wild-type mouse (arrowheads). In some cells, internalized FITC-albumin is localized close to the basal aspect of the cell (arrows). (H) In contrast, 15 min after FITC-albumin injection into a Shank2 knockout mouse, the majority of renal tubules contain no albumin. In a few cells, the injected FITC-albumin appears stuck on the brush border where it aggregates in large vesicles ( , arrowheads). Bar – 20 μ m.

Article Snippet: Kidney sections were blocked (10% normal goat serum and 1% bovine serum albumin [BSA] in PBS) and incubated overnight at 4°C with primary antibody against Rb polyclonal Shank2 (1:100, Santa Cruz; Santa Cruz, CA).

Techniques: Knock-Out, In Vivo, Expressing, Transmission Assay, Electron Microscopy, Membrane, Injection

Journal: Physiological Reports

Article Title: Shank2 Regulates Renal Albumin Endocytosis

doi: 10.14814/phy2.12510

Figure Lengend Snippet: Caveolin-1 expression is altered in Shank2 knockout podocytes. (A) Western blot analysis demonstrates decreased caveolin-1 expression in Shank2 knockout podocytes compared to wild type. Densitometric analysis indicates a 70 ± 6% reduction in caveolin expression in the knockout podocytes (* P < 0.05, n = 3 experiments). (B) Representative immunofluorescence images demonstrate that caveolin-1 is mislocalized away from the cell edge in Shank2 knockout podocytes compared to wild type. Bar – 10 μ m.

Article Snippet: Kidney sections were blocked (10% normal goat serum and 1% bovine serum albumin [BSA] in PBS) and incubated overnight at 4°C with primary antibody against Rb polyclonal Shank2 (1:100, Santa Cruz; Santa Cruz, CA).

Techniques: Expressing, Knock-Out, Western Blot, Immunofluorescence