Immobilon crescendo

Total RNA was isolated using TRIzol regent (Invitrogen) according to the manufacturer’s instructions. For mRNA expression, total RNA was reverse transcribed into cDNA using an oligo 18 T primer, and gene expression levels were then measured by qPCR with a GoTaq^® 2-Step RT-qPCR System for SYBR Green-based detection. The HPRT1 gene was used as a reference. The primer sequences are listed in Table EV1. miRNA expression levels were quantified by miRCURY LNA miRNA PCR Assays (Qiagen) using U6 as a reference, according to the manufacturer’s instructions. All qPCR assays were performed using an Applied Biosystems^® QuantStudio 6 Flex Real-Time PCR System.
Western blotting was performed using standard protocols. Briefly, cells were lysed with RIPA buffer and prepared in 1× sodium dodecyl sulfate (SDS) sample loading buffer, boiled for 10 min at 95 °C, separated on a 10% SDS polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk in TBST for 1 h at room temperature, incubated in primary antibody diluted in TBST overnight at 4 °C, washed three times for 5 min each with TBST, and incubated for 1 h with secondary antibody. Membranes were washed three times for 10 min each with TBST and the chemiluminescence signal was detected by Immobilon^® Crescendo (Millipore) and imaged by ChemiDOC™ MP (Bio-Rad).

Cells lysates were prepared in RIPA buffer, diluted in 1× SDS sample buffer and heated for 10 min at 95°C. The total proteins were separated on 10% SDS‐PAGE gel and transferred to a PVDF membrane. Membrane was blocked with 5% milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, followed by HRP‐conjugated secondary antibody for 1 h at room temperature. In between steps, membrane was washed three times in TBST for 10 min each. The HRP activity was detected by Immobilon® Crescendo (Millipore) and images were acquired by ChemiDOC™ MP (Bio‐Rad).