These assays were performed as described previously [43 (link), 59 (link)]. The gene-specific primers are as follows: for mouse Atp6v1c1, we used the same primers as described in [18 (link), 43 (link)]; for β-actin (expected product of 517 bp) sense 5’-CATTGAACATGGCATTGTTACC-3’ and antisense 5’-CAGCTCATAGCTCTTCTCCAGG-3’; for human ATP6V1C1 (common primers for C1-a and C1-b; expected product C1a: 219bp; C1b: 165bp) sense: 5’-ATGACTGAGTTCTGGCTTATATC-3’ and antisense: 5’-AGCTACTTTCTTAACCACTCC-3’; for human ATP6V1C2 (common primers for C2-a and C2-b; expected product C2a: 487bp; C2b: 349bp) sense: 5’-CGAATCTCTCTCAGACATGG-3’ and antisense: 5’-CTGGAAGTTCACTGGTAGTCC-3’. Anti-Atp6v1c1 (H-300) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA.). Antibodies to mTOR, phospho-T398 p70 S6 kinase, p70 S6 kinase, phospho-S473 Akt, Akt, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) and p44/42 MAPK (Erk1/2) from Cell Signaling Technology (Danvers, MA). Anti-tubulin (E7) and anti-LAMP-1 (1D4B) were from Developmental Studies Hybridoma Bank (DSHB). All assays were repeated three times.
Free full text: Click here
