Horseradish peroxidase conjugated secondary antibody

Cells were lysed, resolved by SDS-PAGE, and transferred to nitrocellulose filter membranes (Biorad, Milan) [27 (link)]. After blocking, membranes were incubated with: anti-PD-L1 (R&D Systems, Milan, Italy), -pSTAT3, -STAT3, -pERK1/2 and -ERK1/2 (all from Cell Signaling Technology, Danvers, MA). After incubation with horseradish peroxidase-conjugated secondary antibody (PerkinElmer, Milan, Italy), reaction was visualized with ECL using ImageQuant LAS4000 (GE Healthcare, Milan, Italy).

Cells were treated with various concentrations of SK228 for 24 or 48 h. After treatment, cells were collected and lysed in PRO-PREPTM Protein Extraction solution (iNtRON Biotechnology). The lysates were incubated on ice for 20 min and centrifugated at 13,000 g for 30 min. Protein concentrations were measured by using protein assay reagents (Pierce). Equal amounts of total protein were mixed with 4X sample loading buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 20% SDS, 2.5% bromophenol blue, and 5% 2-mercaptoethanol), boiled for 10 min and then fractionated by using electrophoresis on 8% or 10% SDS-PAGE. A pre-stained protein ladder (Fermentas) was used as the molecular weight standard. Proteins were electrically transferred to PVDF membranes and treated for 1 h at room temperature with 0.05% TBST and 5% non-fat milk. Blots were subsequently incubated at 4°C overnight with primary antibodies. Immunoreactive proteins were detected after incubation with horseradish peroxidase-conjugated secondary antibody (PerkinElmer) for 1 h at room temperature. The immunoblots were visualized by using enhanced chemiluminescence (Perkin Elmer). All antibodies used are provided in Table S1.

Treated cells were lysed with RIPA buffer containing 10 µg/ml of protease inhibitor (Sigma), and then the lysate was centrifuged at 10,000×g for 15 min at 4°C and the supernatant collected for immunoblotting. The protein concentration was measured by the Bradford assay, and samples containing 20 µg of protein were separated by 10 or 12% SDS–PAGE, and then transferred to Immobilon-P membranes for 2 h at 200 V (Millipore) in a Trans-Blot Electrophoretic Transfer cell. The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA. After washing for 30 min at RT with PBST, the membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibody (Perkin-Elmer, Boston, MA; 1∶5000 dilution in PBST), then bound antibody was detected using the ECL Western blotting reagent (Amersham), chemiluminescence being detected using a Fuji Medical X-ray film (Tokyo, Japan) and quantified by gel image analyses with Image Pro software. The intensity of the band of interest was divided by that for β-actin or GAPDH (loading controls) and this value normalized to that seen with no treatment.