Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Rabbit anti-INF2 , IF: 1:100 , Bethyl Lab , A303-427A.

Techniques: Western Blot, Immunoprecipitation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: FSGS-Causing INF2 Mutation Impairs Cleaved INF2 N-Fragment Functions in Podocytes

doi: 10.1681/ASN.2019050443

Figure Lengend Snippet: SIM demonstrates that INF2 expression and localization are altered in diseased glomeruli with a R218Q mutation. Kidney sections from normal individuals (A and B) and INF2 R218Q–associated FSGS (C and D) were stained for synaptopodin (gray), INF2 N-terminal region (N, red), INF2 C-terminal region (C, green), and nuclei (blue). Two representative glomeruli from two normal individuals and two diseased glomeruli from a single patient with R218Q INF2–mediated FSGS are shown. (i and ii) SIM-processed low-magnification micrographs. (i) Maximum intensity projection of 3D z-stack optical frames. Scale bar, 20 μm. (ii) A representative single optical frame of the 3D z-stack shown (i). Blue boxes highlight region of interest (ROI) used for colocalization analysis. Scale bar, 20 μm. (iii) Colocalization analysis of INF2 with synaptopodin. Scale bar, 2.5 μm. White boxes highlight foot process structures. INF2 N-terminal and INF2 C-terminal antibody staining patterns were differentially localized. (iv) Colocalization and corresponding intensity profiles of INF2 and synaptopodin in foot process structures. Scale bar, 0.7 μm. INF2 N-terminal staining colocalized with synaptopodin in normal foot process structures (A-iv and B-iv). This colocalization is lost in effaced regions of R218Q INF2 samples (arrow highlights; C-iv and D-iv). Intensity profiles confirm colocalization of fluorescent signal intensities. (E) Overlay of INF2 N- and C-terminal staining shown in (B-i and D-i). Green boxes highlight ROI used for C-terminal staining analysis. Scale bar, 20 μm. C-terminal staining in cell body remains unaltered with the disease mutation. Additional INF2 N- and C-terminal staining of normal and disease glomeruli collected with confocal microscopy are shown in Supplemental Figure 3. (F) Colocalization analysis of INF2 with synaptopodin in noneffaced regions of R218Q disease glomeruli shown in (Ci). Red box highlights ROI used for colocalization analysis. Colocalization and corresponding intensity profiles of INF2 and synaptopodin in foot process structures. Scale bar, 0.7 μm. Colocalization of INF2 N-terminal staining with synaptopodin in foot process structures was lost in noneffaced regions of R218Q disease glomeruli as well.

Article Snippet: The following antibodies were used for immunofluorescence analysis: cortactin (ab333333, 1:50; Abcam), KDEL (ER-marker) (ab176333, 1:50; Abcam), mDIA (610848, 1:50; BD Biosciences), INF2 N terminus (SAB1401801, 1:50; Sigma-Aldrich), INF2 C terminus (20466–1 AP, 1:50; Proteintech), and synaptopodin (AP33487SU, 1:50; Origene).

Techniques: Expressing, Mutagenesis, Staining, Confocal Microscopy