Mbp antibody

Immunofluorescence staining was performed the same way as previously implemented [30 (link)]. Firstly, after anesthetizing, the mouse was perfused through ice-cold PBS (0.1 mol/L, 20 mL) via its cardiac apex, followed by another perfusion with 4% paraformaldehyde (PFA). Secondly, the whole brain was extracted and immersed by 4% PFA overnight and was thereafter immersed in 30% sucrose at 4 °C for 72 h. Thirdly, for subsequent experiments, the brains were cut into coronal slices (8 μm) and fixed onto glass slides. After blocking by 5% BSA and 0.3% Triton X-100, these coronal slices were then incubated with primary antibodies overnight at 4 °C. The information of antibodies was as follows: SLC45A3 antibody (1:100, Abcam, ab137065) and MBP antibody (1:500, Cell Signaling Technology, CST#83683S). Fourthly, the sections were incubated by secondary antibodies at 37 °C for 2 h. The secondary antibodies were Alexa Fluro 488-conjugated donkey anti-mouse IgG (1:500, Invitrogen, A21202) and Alexa Fluro 555-conjugated donkey anti-rabbit IgG (1:500, Invitrogen, A32794). Finally, for each mouse, 5 sample slices were examined, and 3 fields of view were chosen per slice to calculate the mean number of target cells by a fluorescence microscope (Leica, Mannheim, Germany).

Clone the full-length cDNA of OsSAUR33 and OsSnRK1A into pGEX-6p-1 and pMAL-c2 vector, respectively. Transform constructs into the E. coli strain BL21 (TSV-A09, Tsingke, PRC) to produce the GST-OsSAUR33 and MBP-SnRK1A fusion proteins. For pull-down, 15 μg SnRK1A-MBP or MBP was incubated with MBP-Tag Dextrin Resin (ATSSE0401, Abbkine, USA) at 4 °C for 2 h then 40 μg OsSAUR33-GST was added and incubated overnight at 4 °C. The beads were washed three times with PBS (SL6110, Coolaber, PRC) + 1% Triton 100 (CT11451, Coolaber, PRC) and three times with PBS, then were boiled at 95 °C for 10 min with 5× sodium dodecyl sulfate (SDS) loading buffer (SL1180, Coolaber, PRC). For western blotting, the GST antibody (Cell signaling technology, Danvers, MA, USA) and MBP antibody (Cell signaling technology, Danvers, MA, USA) were used to detected the proteins.