Journal: bioRxiv

Article Title: A Novel TRPC6 Mutation Causes Autosomal Dominant FSGS

doi: 10.1101/2025.02.11.637765

Figure Lengend Snippet: iPS-cell derived podocytes have reduced expression of slit diaphragm proteins, ER congestion, and reduced lysosomes. Panel A shows representative immunoblots of podocyte markers including Nephrin, Synaptopodin, TRPC6, Podocin, CD2AP, GLEPP1, and WT1, the housekeeping protein GAPDH. Panel B shows the quantification of immunoblots for the podocyte markers. Data represents three biologically independent experiments and is defined as ± standard error of the mean. We performed one-way ANOVA with Dunnett’s multiple comparison post hoc analyses with a family-wise alpha threshold of 0.05. Panel C shows electron micrograph of differentiated podocytes with protein aggresomes in the ER of Duke 5 and Duke 7 podocytes. Panel D shows LAMP1 vesicles, Podocin, and TRPC6 expression in podocytes. The LAMP1 vesicle number remains unchanged in the podocytes. Panel E shows lysosomal biogenesis-associated RAB7 expression in the podocytes. RAB7 expression was reduced in the mutants. Data is representative of three biologically independent experiments and the data is defined as ± standard error of the mean. We performed one-way ANOVA with Sidak’s multiple comparison post hoc analyses with a family-wise alpha threshold of 0.05. Scale bar: Panel C, top row 1000 µm, bottom row 500 nm; Panel D, top row 20 µm, bottom row 5 µm; Panel E, 100 µm.

Article Snippet: The primary antibodies used included guinea pig Anti-Nephrin (ARP; #GPN02, 1:500), rabbit Anti-Podocin (Abcam, mAb #ab50339, 1:1000), rabbit Anti-TRPC6 (Abcam, pAb #ab228771) and mouse Anti-CD2AP (B-4) (Santa Cruz, #sc-25272), Calnexin (C5C9) Anti-Rabbit (Cell Signaling, mAb #2679), LAMP1 (D2D11) XP Anti-Rabbit (Cell Signaling, mAb #9091), RAB7 (D95F2) XP and Anti-Rabbit mAb (Cell Signaling, mAb #9367).

Techniques: Derivative Assay, Expressing, Western Blot, Comparison