Gfra1 antibody

Cell slides or smears were prepared and then fixed with 4% paraformaldehyde for 20 min at 4 °C. Nonspecific binding sites were blocked with 10% BSA/PBS for 60 min at room temperature, followed with 0.1% TritonX-100 permeable treatment for 10 min. Sections were incubated with the TFEB antibodies (1:200 dilution; Santa cruz), GFRA1 antibody (1:200 dilution; Santa cruz) overnight at 4 °C. Then, fluorescence-labeled secondary antibodies (donkey anti–rabbit Alexa Fluor 488, donkey anti–mouse Alexa Fluor 555, 1:500 dilution; Jackson ImmunoResearch) were used. Nuclei were counterstained with DAPI (Sigma-Aldrich). The fluorescence signals were detected under a laser scanning confocal microscope (Carl Zeiss LSM-510, Germany) equipped with an argon laser (488 nm), a He/Ne laser (543 nm), an EC Plan-NEOFLUAR 63×/1.25 objective and a LD LCI Plan-APOCHROMAT 25×/0.8 objective (Zeiss). Digital images were taken and processed using Aim software (Zeiss Systems).

Protein was extracted from GmGSCs-I-SB cells transduced with Lin28a or control by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime). Then, total cell protein was resolved by SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane. The membranes were incubated with primary antibodies, including the LIN28A antibody (1:500, Santa Cruz), SOX2 antibody (1:200, Bioss), PCNA antibody (1:1000, Biolegend), OCT4 antibody (1:200, Bioss), ETV5 antibody (1:1000, abcam), GFRA1 antibody (1:400, Santa Cruz), ERK antibody (1:1000, Cell Signaling Technology), p-ERK antibody (1:500, Cell Signaling Technology), AKT antibody (1:1000, BOSTER, Wuhan, China), p-AKT antibody (1:500, Sangon), S6 antibody (1:1000, Cell Signaling Technology), mTOR antibody (1:1000, Cell Signaling Technology), p-mTOR antibody (Ser2448, 1:1000, Cell Signaling Technology), pS6 (Ser235/236, 1:1000, Cell Signaling Technology), CXCR4 antibody (1:400, Bioss), and anti-β-actin antibody (1:1,000, Cell Signaling Technology). Horseradish peroxidase-conjugated anti-rabbit or mouse IgG antibodies were used as secondary antibodies (1:1000, BOSTER). Detection was performed using the Chemistar Hiht-sig ECL Western Blotting Substrate (Tanon). The results were analyzed by Tanon-410 automatically image system (Shanghai Tianneng Corporation, China).