Anti-RPS6A-P237 | Phosphorylated (Ser237) 40S ribosomal prot

Left panel is developed with anti-RPS6-Ser237,  while right panel anti-RPS6, which serves as a loading control.

1 and 4 - 0.5 µg of Arabidopsis thaliana whole leaf extract from untreated leaf discs

2 and 5 - 0.5 µg of Arabidopsis thaliana whole leaf extract from leaf discs treated with 10 mM glutamine (TOR activator) for 8 hours

3 and 6 - 0.5 µg of Arabidopsis thaliana whole leaf extract from leaf discs treated with 2 µM AZD-8055 (TOR inhibitor) for 8 hours

Courtesy of Dr. Brendan O'Leary, University of Western Australia, School of Molecular Sciences, ARC Centre for Plant Energy Biology, Australia

20 µg/well of total protein freshly extracted freshly from 16 d old Arabidopsis thaliana seedlings total leaf of wildtype (1) and mutant (2) with 2x SDS loading dye exact buffer and denatured at 80 °C for 5 min.were separated on 12% SDS-PAGE  and blotted 10 min to PVDF (pore size of 0.2 um), using using Turbo transfer (BioRad). Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in TBS-T ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly once, then washed 5 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:15 000 in  for 1h/RT with agitation. The blot was washed as above and reaction was visualized with chemiluminescence, following manufacture's instructions. Exposure time was 5 seconds.

Courtesy of Dr. Arsheed Sheikh, King Abdullah University of Science and Technology (KAUST), Saudi Arabia