Anti actin ac 40

BMDM were washed three times with 2 ml of ice-cold PBS and lysed with 0.15 ml of M-PER mammalian protein extraction reagent with Halt protease and phosphatase inhibitors (Thermo Scientific). Equal amounts of protein per sample/lane were denatured and resolved by SDS-PAGE. Proteins were transferred to Immobilon-FL membranes (Millipore, Billerica, MA), and quantitative immunoblotting was performed using the Odyssey infrared immunoblotting detection system (LI-COR Biosciences, Lincoln, NE). Primary antibodies used were anti-CaMKK2 (6/CaM Kinase, 610544; BD Biosciences; dilution 1:1000); anti-actin (AC-40, catalog # A4700; Sigma-Aldrich; dilution 1:20000); anti-phospho-CaMK1 (Thr¹⁷⁷; PA5-38434 ThermoFisher, dilution 1:500), anti-phospho-AMPKα (Thr¹⁷², 40H9; catalog # 2535; dilution 1:1000) and AMPKα (F6, catalog # 2793; dilution 1:1000) were from Cell Signaling (Danvers, MA). Secondary antibodies used were anti-mouse IgG Alexa Fluor 680 (catalog # A28183; Invitrogen; dilution 1:1000) and anti-rabbit IgG IRDye800-conjugated antibody (catalog # 611-132-002; Rockland Immunochemicals, Gilbertsville, PA; dilution 1:5000). All antibodies were used according to the manufacturer’s instructions. Images of uncut and unmanipulated blots are presented in Supplementary Fig. 12.

Cells were harvested directly into lysis buffer (150 mM NaCl, 1 % Nonidet P-40, 50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, protease inhibitor cocktail) and 20 μg of total protein in NuPAGE LDS Sample Buffer (Life Technologies, USA) loaded on 10 % polyacrylamide gels, separated and transferred onto nitrocellulose membranes in Mini-PROTEAN Electrophoresis System (Bio-Rad, USA) for 90 min applying 100 V in transfer buffer (240 mM Tris, 190 mM glycine, 20 % methanol). Membranes were blocked in 5 % non-fat milk in PBS with 0.1 % Tween and incubated overnight with primary antibodies at 4 °C. Primary antibodies used: anti-p63 clone SFI-6 (DCS Innovative Diagnostik-Systeme, Germany), anti-TAp63 clone TAp63-4.1 and anti-ΔNp63 clone ΔNp63-1.1 and polyclonal ΔNp63-44 [4 (link)] and anti-actin AC40 (Sigma-Aldrich, USA). Membranes were then incubated with appropriate HRP-conjugated secondary antibodies (RAM-Px, SWAR-Px, Dako, Denmark) diluted 1 : 1000 for 1 h at room temperature. Signal was detected using ECL reagent (Amersham Pharmacia Biotech, UK).

The following reagents were used in this study: Anti SCRIB C-20 (Goat, Santa Cruz Biotechnology, Dallas, TX), Anti-SPTBN1 and E-cadherin (Mouse, BD Transduction Laboratories, San Jose, CA), Anti-SPTBN2 A301-117A (Rabbit, Bethyl, Montgomery, TX), Anti-GFP HRP ab6663 (Abcam, Cambridge, MA), Anti-FLAG HRP clone M2 (Sigma, Saint Louis, MO), Anti-pericentrin PRB-432C (Rabbit, Covance, Princeton, NJ), Anti-Actin AC-40 (Sigma). SiRNA sequences: Non-Targeting control siRNA (luciferase) (UAAGGCUAUGAAGAGAUAC), SPTBN1 (UGAUGGCAAAGAGUACCUCTT), SCRIB-ORF1 (GCACUGAGGAGGAGGACAATT), ORF2 (GAACCUCUCUGAGCUGAUCTT), UTR1 (GUUCUGGCCUGUGACUAACTT) and UTR2 (GGUUUAAGGAGAAUAAAGUTT) were ordered at Eurofins (France).