Bronectin

For western blotting, mouse kidney tissues were lysed in 300 μL of cell lysis buffer containing 150 mM NaCl, 1 % IGEPAL â CA-630, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8.0), and a protease inhibitor cocktail (Thermo Fisher Scienti c). Kidney tissue lysates were resolved by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto polyvinylidene uoride membranes (Millipore), blocked with 5 % skim milk, and incubated with primary antibodies against E-cadherin (1:1000; Santa Cruz), collagen (1:500; Santa Cruz), α-SMA (1:10000; R&D), bronectin (1:1000; Santa Cruz), TGF-β1 (1:1000; Santa Cruz), and GAPDH (1:1000; Cell Signaling Technology). The membranes were then washed three times with 1´ PBS with Tween-20 (AMRESCO â ) for 5 min, incubated with horseradish peroxidase-conjugated secondary antibodies, and washed again using the same procedure. Target proteins were visualized using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences), and band density was measured using NIH Image J software.

For western blotting, mouse kidney tissues were lysed in 300 μL of cell lysis buffer containing 150 mM NaCl, 1 % IGEPAL â CA-630, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8.0), and a protease inhibitor cocktail (Thermo Fisher Scienti c). Kidney tissue lysates were resolved by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto polyvinylidene uoride membranes (Millipore), blocked with 5 % skim milk, and incubated with primary antibodies against E-cadherin (1:1000; Santa Cruz), collagen (1:500; Santa Cruz), α-SMA (1:10000; R&D), bronectin (1:1000; Santa Cruz), TGF-β1 (1:1000; Santa Cruz), and GAPDH (1:1000; Cell Signaling Technology). The membranes were then washed three times with 1´ PBS with Tween-20 (AMRESCO â ) for 5 min, incubated with horseradish peroxidase-conjugated secondary antibodies, and washed again using the same procedure. Target proteins were visualized using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences), and band density was measured using NIH Image J software.