Image studio software version 4

Cortical tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma) supplemented with protease (Complete Mini, Roche, Basel, Switzerland) and phosphatase (PhosStop, Roche) inhibitors according to standard protocols. Western blot analyses were conducted according to standard protocols. Briefly, soluble extracts were loaded onto Criterion, 4%–15% Tris-HCI 4 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Berkeley, CA, USA), separated at 120 V, and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) at 30 V for 2 h or overnight at 4 °C. Membranes were blocked with 5% BSA/1X TBS-T (Tris-buffered saline with 0.1% Tween 20) for 1 h at room temperature, and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, and secondary antibodies (1:5000 dilution; IR-Dye antibodies, LI-COR, Lincoln, NE, USA) for 1 h at RT. Membranes were washed in 1X TBS-T and scanned using the Odyssey Infrared Imaging System (LI-COR). Primary antibodies were used as follows: goat anti-Gli3 (1:500; R&D Systems) and α-Tubulin (1:5000, Abcam). Quantification and analysis were conducted using the Odyssey System and Image Studio Software version 4.0.21 (LI-COR, Lincoln, NE, USA).

After dissection, tissues were kept at –80 °C till use. To prepare protein lysates, tissues were homogenized with an Ultra-Turrax T10 (VWR) in 10 volumes (w/v) of ice-cold modified RIPA buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) supplied with Complete Proteinase Inhibitor Cocktail tablets (1873580, Sigma-Aldrich) and PhosSTOP phosphatase Inhibitor Cocktail tablets (0490683701, Sigma-Aldrich). After a further 5-min sonication step in an ultrasonic bath for shearing genomic DNA, the lysates were centrifuged at 16,200× g and 4 °C for 20 min to obtain the soluble protein.
Western blot analyses were performed as described earlier [44 (link)]. Primary antibodies were diluted in TBS-T (TBS with 0.1% Tween 20) and the respective dilution factors are summarized in Table A1. For total protein detection, membranes were incubated with SYPRO Ruby Protein Blot Stain (Thermo Fisher Scientific) according to the manufacturer’s protocol, prior to blocking. For caspase-3 analyses, a commercial sample of cell extracts treated with cytochrome c served as positive control (9663, Cell signaling). All fluorescence signals were detected and quantified using the ODYSSEY FC Imaging System with Image Studio software version 4.0 (both LI-COR Biosciences, Bad Homburg, Germany).