Anti β actin

For whole cell lysates, proteins were harvested from PBS-washed cells using mild lysis buffer (150mM sodium chloride, 1% NP-40, 50mM Tris pH 8.0) supplemented with protease inhibitor cocktail (Sigma-Aldrich, Inc.). Proteins from supernatants were collected using cold-acetone precipitation followed by centrifugation at 14,000g for 10 min. Both mixtures were incubated on ice for 15 min and centrifuged at 16,000g for 15 min. Supernatant and whole cell lysate protein concentrations were calculated by the BCA method. 25μg of protein extracts were run on SDS-PAGE gels, transferred to nitrocellulose and incubated with following antibodies diluted at 1:500–1000 ratio in 5% BSA solution: anti-FLAG (DYKDDDDK (FG4R)-MA1-91878, Thermo fisher), anti-Shh-N (AF464,R&D), anti-β-actin (Sigma A3854). Blots were incubated with secondary antibody-conjugated HRP and developed with ECL western blotting detection reagents as described elsewhere.^{(59 (link))} Probed membranes were stripped with Reblot strong stripping solution (Millipore) for 15mins, washed, re-blocking with 5% skim milk, then reprobed with anti-β-actin for normalization. For supernatant normalization, membranes were ponceau S stained before blocking.