Chemismart5000 digital imaging system

Protein lysates were separated by SDS-PAGE and subsequently transferred on nitrocellulose membranes by Western blotting. Membranes were incubated with indicated primary antibodies overnight at 4°C according to manufacturer’s instructions. Antibodies are listed in Supplementary Methods. Chemiluminescent reactions were processed using Femto-Glo ECL reagents (PJK) and documented on a ChemiSmart5000 digital imaging system (Vilber-Lourmat). Proper loading controls (GAPDH or β-actin as indicated) were examined on the same membrane. Images were quantified using Bio1D software (Vilber-Lourmat).

For Western blotting, equivalent amounts of sample protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Macherey Nagel, Düren, Germany). After blocking with 5% BSA in TBS-T (20 mM Tris pH 7.6, 150 mM NaCl and 0.02% Tween-20), the membrane was immunoblotted with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit anti-LRRC8A (1 μg/mL, [4 (link)] kindly supplied by T. J. Jentsch); rabbit anti-p-ULK (1:1000, Cell Signaling, Frankfurt am Main, Germany; #14202); rabbit anti-ULK (1:1000, cell signaling, #8054); rabbit anti-p-Akt^Ser473 (1:1000, Cell Signaling; #4060); rabbit anti-Akt^pan (1:1000, Cell Signaling; #4685); and rabbit anti-GAPDH (1:2500, Cell Signaling; #2118) antibodies. The corresponding peroxidase-labeled secondary antibody (1:10,000, Jackson ImmunoResearch, Ely, UK) was used. Signals were detected using an enhanced chemiluminescence reagent (HRP juice; PJK GmbH, Kleinblittersdorf, Germany) and a ChemiSmart5000 digital imaging system (Vilber-Lourmat, Collégien, France). Densitometrical quantification was performed with the Fiji software [64 (link)].

Protein lysates were separated on 10% SDS-PAGE gels. Gels were transferred to PVDF membranes by Western blotting. Membranes were blocked in 0.1% TBS-T containing 3% w/v BSA for 1 h at RT, washed three times in 0.1% TBS- T and incubated with indicated primary antibodies (GAPDH #2118; phospho-SMAD1/5 (Ser463/465) #9516; SMAD1 #6944; phospho-SMAD2 (Ser465/467) #3108, SMAD2 #3122, Cell Signaling Technology) overnight at 4 °C following manufacturer’s instructions. Secondary HRP conjugated antibodies goat-anti-mouse or goat-anti-rabbit IgG (Dianova) were used. Chemiluminescent reactions were processed using WesternBright Quantum HRP substrate (advansta) and documented by using a ChemiSmart5000 digital imaging system (Vilber-Lourmat).