Fig. 7. Afadin is phosphorylated by S6K1 in Tsc1-mutant conditions.

a Cells were serum starved overnight and subsequently amino acid starved with EBSS (Earle’s Balanced Salt Solution) for 1.5 h with or without the indicated treatments. They were then collected and cell lysates were probed with the indicated antibodies. Asterisk represents non-specific cross-reactive bands. b Densitometry analysis and relative quantification of the phosphorylated forms of the indicated proteins. Actin was used as a loading control. Mean ± SEM. n = 4 independent experiments were analyzed. c Western blots of mouse whole-kidney extracts at pre-cystic age (post-natal day 20), randomly fed, and probed with the indicated antibodies. d Densitometry analysis and relative quantification of the phosphorylated form of Afadin. Actin was used as a loading control. Mean ± SEM. n = 3 Tsc1 f/f, n = 3 Tsc1 f/f; S6k1 −/−, n = 4 Ksp-Cre; Tsc1 f/f, and n = 4 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were analyzed. e Western blot of protein lysates extracted from patient samples. Renal tumors (t) or normal tissue adjacent to renal tumor (n) were collected from TSC patients and non-TSC patients. Protein lysates then probed with the indicated antibodies. f Densitometry analysis and fold change of TSC patients over non-TSC patients of the indicated proteins relative to the indicated loading control. Mean ± SEM. n = 5 TSC patients, n = 7 non-TSC patients were analyzed. g Analog-sensitive S6K1 was co-transfected in Tsc1/S6k1 dKO mIMCD3 cells with FLAG-Afadin-WT. An in vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S both in presence and in absence of 20 nM rapamycin. After immunoprecipitation using an anti-FLAG antibody, the thio-phosphorylation of Afadin was revealed by western blot using an anti‐thiophosphate ester antibody. Expression levels of Afadin, phospho-S1795-Afadin, FLAG and phospho-RPS6 were revealed by western blot on total extracts using the indicated antibody. Source data are provided as a Source Data file.