Anti hnrnpc

Cells were collected and lysed in RIPR buffer (Beyotime) supplemented with the protease inhibitor cocktail (Roche). Protein concentrations were quantified using the BCA protein assay (Pierce). The cell lysate containing 30 μg total protein was heat‐denatured and subjected to SDS–PAGE, followed by transferring to PVDF membrane. The membrane was incubated with the primary and secondary antibodies and visualized with SuperSignal West Pico Chemiluminescent HRP substrate (Thermo). The anti‐HNRNPC, anti‐PKR, anti‐beta‐actin, and anti‐alpha‐tubulin antibodies were purchased from Abcam. Nuclear and cytoplasmic fraction proteins were separated by Nuclear/Cytosol Fractionation Kit (Biovision, K266‐25). The density quantifications were analyzed by ImageJ software.

RIP assay was performed by employing Z-Magna RIP™ RNA-binding Protein Immunoprecipitation kit (Millipore, Bedford, MA, USA). RIP lysis buffer was added to lyse HNSCC cells. Afterward, cell lysates were co-cultivated with anti-IgG (Abcam), anti-Ago2 (Abcam) or anti-HNRNPC (Abcam). Eventually, the enrichment of TM4SF19-AS1, miR-153-3p and LAMC1 was examined by RT-qPCR analysis. The independent assay was performed three times.