Protein loading buffer

The DNA sequence of LHFPL3-AS1-long or LHFPL3-AS1-short was amplified with sequence-specific primers containing T7 RNA polymerase promoter sequence. Then the purified PCR product was used as the template for in vitro transcription. The RNA transcript was synthesized using T7 in vitro transcription kit (Promega, USA) and biotinylated with EZ-Link Biotin kit (Thermo scientific, USA) according to the manufacturer’s instructions. The biotin-labeled RNAs were purified with mirVana miRNA Isolation Kit (Ambion, USA).
Cancer stem cells (5 × 10⁶) were lysed using immunoprecipitation lysis buffer (Beyotime, China) containing 2 mM protease inhibitor. After centrifugation at 300×g for 5 min, the cell lysate was incubated with the biotinylated sense or antisense LHFPL3-AS1 RNA at 4 °C overnight. Subsequently, the mixture was incubated with streptavidin-conjugated Dynabeads (Thermo Scientific, USA) on a rotator for 2 h at 4 °C. The beads were washed with lysis buffer and then boiled in protein loading buffer (Sangon Biotech, Shanghai, China). The proteins were separated by gel electrophoresis and stained with Coomassie brilliant blue (Beyotime, China). The separated proteins bands specific for the sense LHFPL3-AS1 RNA were excised for mass spectrometry analysis.

Cx50WILD-transfected, Cx50V44A-transfected, EGFP-transfected HeLa cells and human retinal epithelial cells (ARPE-19, ATCC) were seeded in 100-mm dishes for 24 h. ARPE-19, which has been reported to express Cx43 [10] (link), served as a control. The cells were collected in ice-cold PBS and were then centrifuged at 1000 rpm at 4°C for 5 min. After removing the supernatant, total protein was extracted from the cells with a lysis buffer (Sangon Biotech, Shanghai, China). After incubation on ice for 30 min, the extracts were centrifuged at 14,000 rpm at 4°C for 15 min. Then, the supernatant of each sample was transferred to a fresh tube and boiled with a protein-loading buffer (Sangon Biotech). The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes and blotted with anti-Cx43 rabbit polyclonal antibody (1∶200 dilution, Santa Cruz) and anti-GAPDH rabbit monoclonal antibody (1∶5000 dilution, Cell Signaling Technology, USA). After incubation in fluorescent secondary antibodies (1∶5000 dilution, Cell Signaling), the blots were analyzed using the Bio-Rad ChemiDoc MP imaging system. The target protein expression levels were normalized relative to GAPDH expression.