Sox2 antibody

Whisker and pelage DP spheres were isolated according to the above methods. After isolation, DP spheres were fixed with 4% paraformaldehyde for 20 mins, washed and centrifugated with TBST three times, then permeabilized with 0.1% Triton X-100 for 30 min at room temperature. After blocking with 5% BSA for 1 h, 1:200 dilution of Sox2 antibody (Bioss) is carried out overnight at 4 °C and subsequently incubated with a 1:200 dilution of 488/594-conjugated anti-rabbit IgG antibody (Abcam) for 2 h, washed and centrifugated with TBST three times. DAPI was incubated for 30 min, washed and centrifugated with TBST three times, finally prepared to image with a confocal laser scanning microscope.

GmGSCs-I-SB-GFP cells and GmGSCs-I-SB-Lin28a cells were fixed in 4% paraformaldehyde (PFA) for 15 min and treated with 0.1% Triton X-100 for 10 min at room temperature55 (link). Then, the samples were blocked with 1% BSA for 30 min and incubated in primary antibody at 4 °C overnight. The following primary antibodies were used: LIN28A antibody (1:200, Santa Cruz), SOX2 antibody (1:100, Bioss), OCT4 antibody (1:100, Bioss), PLZF antibody (1:100, Bioss), and GFRA1 antibody (1:100, Bioss). Goat anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 (1:500; Invitrogen) were incubated for 1 h at room temperature in the dark. Cell nuclei were stained by Hoechst 33342 (Sigma) at room temperature (RT) for 5 min. The samples were washed by PBS three for 5 min. Images were analyzed by a EVOS^® Imaging System.

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).