Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: Impacts of overexpression and neutralization of FSTL1 on renal fibrosis and on the canonical Wnt/β-catenin pathways in UUO kidneys. A – C , effects of FSTL1 overexpression on renal fibrosis and on the Wnt/β-catenin pathway. Male mice at 8 weeks of age were subjected to UUO on left ureters. FSTL1-HA or pcDNA3.1 plasmid was injected via the tail vein on day 2 after UUO surgery. Mice were sacrificed the next day, and right (Ctrl) and left (UUO) kidneys were collected for analysis. A , kidney lysates were analyzed by Western blotting for FSTL1 expression and the fibrosis markers α-SMA, fibronectin-1 (Fn-1), and collagen 1 (Col IA1) ( left panel ). Levels of α-SMA, Fn-1, and Col IA1 relative to GAPDH are presented ( right panels ). B , immunofluorescence for α-SMA and Fn-1 and Masson’s trichrome staining (MTS) on sections from the UUO kidneys of mice injected with pcDNA3.1 or FSTL1-HA plasmids. Frozen sections were used for immunofluorescent staining for α-SMA ( green ) and Fn-1 ( red ). Paraffin sections were used for MTS. The fibrotic blue area was quantified. C , kidney lysates were analyzed by Western blotting for the Wnt/β-catenin pathway (p-LRP6, p-GSK3β, active β-catenin, and β-catenin) ( left panel ). Levels of p-LRP6 relative to LRP6; p-GSK3β relative to GSK3β; and active β-catenin and β-catenin relative to GAPDH are presented ( right panels ). D , effects of neutralization of FSTL1 on the Wnt/β-catenin pathway. Male mice at 8 weeks of age were subjected to UUO on left ureters. Normal goat IgG or goat anti-FSTL1 antibody was injected (i.p.) at 5 μg/g body weight 7 days after UUO surgery. About 6 h later, kidney samples were harvested: kidney lysates were analyzed by Western blotting for p-LRP6, active β-catenin, and β-catenin ( left panel ). Levels of p-LRP6 relative to LRP6 and active β-catenin relative to β-catenin and GAPDH are presented ( right panels ). ∗∗ p < 0.01; ∗∗∗ p < 0.001. FSTL1, follistatin-like 1; HA, hemagglutinin; IgG, immunoglobulin G; LRP6, low-density lipoprotein receptor–related protein 6; α-SMA, alpha-smooth muscle actin; TGF-β, transforming growth factor beta; UUO, unilateral ureteral obstruction.

Article Snippet: Membranes were probed with anti-Wnt1 (catalog no.: ab15251; Abcam), anti-FSTL1 (catalog no.: AF1738; R&D Systems), anti-V5 (catalog no.: 13202; Cell Signaling Technology), anti-HA (catalog no.: 05-904; Millipore), anti-WNT3a (catalog no.: 2721; Cell Signaling Technology), anti-Myc (catalog no.: ab9106; Abcam), anti-SNAP (catalog no.: CAB4255; Thermo Fisher Scientific), anti-p-GSK3β (catalog no.: 9336; Cell Signaling Technology), anti-GSK3β (catalog no.: 9315; Cell Signaling Technology), anti-active (nonphospho) β-catenin (catalog no.: 8814; Cell Signaling Technology), anti-β-catenin (catalog no.: 8480; Cell Signaling Technology), anti-p-LRP6 (catalog no.: 2568; Cell Signaling Technology), anti-LRP6 (catalog no.: 3395; Cell Signaling Technology), anti-α-SMA (catalog no.: ab5694; Abcam), anti-Fn (catalog no.: AB2033; Millipore), anti-collagen 1 (catalog no.: bs-10423R; Bioss Antibodies), anti-TGF-β1 (catalog no.: bs-0086R; Bioss), anti-p-Smad3 (catalog no.: ab52903; Abcam), anti-Smad3 (catalog no.: 9523 or 9513; Cell Signaling Technology), anti-p-Smad1/5/8 (catalog no.: 9511; Cell Signaling Technology), anti-Smad1 (catalog no.: 9743; Cell Signaling Technology), anti-p-JNK (catalog no.: 9251; Cell Signaling Technology), anti-JNK (catalog no.: 9252; Cell Signaling Technology), anti-p-CaMKII (catalog no.: 12716; Cell Signaling Technology), and anti-CaMKII (catalog no.: ab52476; Abcam) antibodies.

Techniques: Over Expression, Neutralization, Plasmid Preparation, Injection, Western Blot, Expressing, Immunofluorescence, Staining