Horseradish peroxidase linked anti rabbit antibody

Samples were lysed in RIPA solution (R0010, Solarbio), centrifuged at 12,000 × g for 30 min at 4°C and mixed with SDS lysates loading buffer and boiled for 5 min at 100°C. Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were incubated with the following primary antibodies: anti-GSK3α/β (sc-7291), anti-phospho-GSK3α/β (Ser21/9) (#9327; Cell Signaling Technology, lnc., Danvers, MA, United States), anti-α-tubulin (sc-398103, Santa Cruz Biotechnology), anti-β-actin (#3700; Cell Signaling Technology, lnc., Danvers, MA, United States) and anti-CD9 (#13403; Cell Signaling Technology, lnc., Danvers, MA, United States) (1:1000). Secondary antibodies were horseradish peroxidase-linked anti-mouse antibody (1:5000; ab205719; Abcam) and horseradish peroxidase-linked anti-rabbit antibody (#7074; Cell Signaling Technology, lnc., Danvers, MA, United States). The membranes were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (WBKLS0500; Merck, Germany).

Protein extracts were prepared as described in [49 (link)]. AMPKα protein expression level was evaluated using a primary antibody directed against AMPKα (Cell signaling technology #2532). This antibody recognizes alpha 1 and alpha 2 isoforms of the catalytic subunit, but not the regulatory beta subunit. 20 μ g of proteins were loaded on the gel and separated by SDS-page. After the transfer onto a PVDF membrane, the membrane was blocked in PBS-Tween 0.1% BSA 5% buffer and then incubated with the primary antibody (1:1000) overnight at 4 °C. After several washes, the membrane was then incubated with a horseradish-peroxidase-linked anti-rabbit antibody (Cell signaling technology #12–2018) for 1 h at room temperature. Signal was detected using PXi (Syngene, Cambridge, UK) after incubation with chemiluminescent substrate (ECL Immobilon Western, WBKLS0100, Merck, Darmstadt, Germany). β-actin (Sigma #A5316, 1:10000, 1 h at room temperature) with an anti-mouse secondary antibody (Cell signaling technology #08–2018; 1:10000, 1 h at room temperature) was used as a loading control for quantification.