Anti lamp 1 1d4b

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.

The following commercially available antibodies were used: anti-Actin (Mab1501R, Millipore), anti-HSP60 (SC-1054, Santa Cruz Biotech), anti-LAMP1 (1D4B, Developmental Studies Hybridoma Bank), anti-P62 (PM045, MBL), anti-LC3B (2775 s, Cell Signaling), anti-c-Myc (C3956, Sigma), anti-Ubiquitin (P4D1, Cell Signaling), anti-PINK1 (75488, Abcam), anti-TBC1D15 (121396, Abcam), anti-PARKIN (15954, Abcam), and anti-MUL1 (HPA017681, Sigma).
For Western analysis, densitometry was done using ImageJ. The intensity of the ubiquitin signal was normalized to that of HSP60, and the average of three separate experiments was taken.