Rabbit anti p gsk 3β

To examine the effect of sLRP6E1E2 on the expression levels of LRP6, β-catenin, pGSK-3β, and Smad 2/3, HDFs were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 in presence of Wnt3a (100 ng/mL) at 50 MOI. At 3 days after transduction, cells were lysed in a solution containing 50 mM Tris-HCl (pH 7.6), 1% Nonidet P-40 (NP-40), 150 mM NaCl, 0.1 mM zinc acetate, and protease inhibitors. Protein concentration was determined by the Lowry method (Bio-Rad, Hercules, CA), and 30 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins from the gels were electrotransferred to polyvinylidene fluoride membrane and initially incubated with primary mouse anti-LRP6 (Santa Cruz biotechnology, Santa Cruz, CA), rabbit anti-β-catenin (Cell Signaling Technology, Beverly, MA), rabbit anti-pGSK-3β (Cell Signaling Technology), rabbit anti-Smad 2/3 (Cell Signaling Technology), or rabbit anti-β-actin antibody (Sigma, St Louis, MO), and then secondarily incubated with the horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). The expression patterns were revealed using enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). And the expression levels of LRP6, β-catenin, pGSK-3β, and Smad 2/3 were semi-quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Ischemic cortex tissue samples were extracted at 3 dpi and homogenized in RIPA lysis buffer. After SDS-PAGE and protein transfer, membranes were incubated with primary antibodies including rabbit anti-IL-17 (1: 1 000, Abcam, Cat# ab79056), mouse anti-Wnt2 (1: 5 000, Abcam, Cat#66656-1-lg), rabbit anti-p-PI3K p85 ((Tyr458)/p55 (Tyr199), 1:1 000, Cell signaling, Cat#17366), rabbit anti-PI3K p-85(1:1 000, Cell signaling, Cat#4257), rabbit anti-p-Akt (Ser473, 1:1 000, Cell signaling, Cat#4060), rabbit anti-Akt (1:1 000, Cell signaling, Cat#4685), rabbit anti-Caspase 3 (1:1 000, Cell signaling, Cat#14220), rabbit anti-β-catenin (1:1 000, Cell signaling, Cat#25362), rabbit anti-p-GSK-3β (Ser9, 1:1 000, Cell signaling, Cat#5558), rabbit anti-GSK-3β (1:1 000, Cell signaling, Cat#12456), and rabbit anti-β-actin (1:3 000, Cell signaling, Cat#5125) overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit or anti-mouse IgG (1: 5 000, Proteintech, USA) for 3 h at room temperature. Bands were visualized with an ECL kit (Thermo).

Immunoblotting was performed as described[38 (link)]. Primary antibodies included rabbit anti-cubilin and sheep anti-megalin (gifts from Prof. Pierre Verroust, INSERM, Paris, France), mouse anti-GSK3β (#610201, BD Biosciences), rabbit anti-pGSK3β (#9336S, Cell Signalling Technology) and guinea pig anti-nephrin (#20R-NP002, Fitzgerald Industries International). Proteins were visualized using enhanced chemiluminescence (Pierce) and the “ChemiDoc XRS” (Bio-Rad). For semi-quantification the QuantityOne software (Bio-Rad) was used, while Coomassie blue staining was used to determine and correct for total protein input.

RIPA lysis buffer that contained a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai) was utilized to lyse cells and lung tissues for thirty minutes on ice. The samples were centrifuged at a centrifugation rate of 12,000 g for thirty minutes to sediment the debris. A BCA Assay Kit (Solarbio Life Science, Beijing, China) was used in order to assess the protein content. Proteins (30μg) were isolated using 10% SDS-PAGE and put into PVDF membranes. These plots were then subjected to incubation overnight at a temperature of 4°C with rabbit anti-α-SMA (1 : 500; Abcam), rabbit anti-S100A4 (1 : 1000; Abcam), rabbit anti-p-ERK1/2 (1 : 1000; Cell Signaling Technology), rabbit anti-fibronectin (1 : 1000; Abcam), rabbit anti-ERK1/2 (1 : 1000; Cell Signaling Technology), rabbit anti-FAK (1 : 1000; Cell Signaling Technology), rabbit anti-p-FAK (1 : 1000; Cell Signaling Technology), rabbit anti-p-GSK-3β (1 : 1000; Cell Signaling Technology), and rabbit anti-GSK-3β (1 : 1000; Cell Signaling Technology). As an internal control, GAPDH (1 : 10000; Abcam) was employed. After the membranes were washed, they were exposed to incubation for one hour at room temperature with HRP-conjugated secondary antibodies. The ImageJ program (version: 1.46) from the National Institutes of Health, Bethesda, MD, was utilized for the purpose of conducting the densitometric analysis.