Nb100 2220

Western blotting was performed using a modified Novus Biological protocol (NB100-2220) with 10%, 12%, 14%, or 4%–20% gradient SDS-PAGE gels. Briefly, cells were collected and lysed with RIPA buffer plus PMSF protease inhibitor. The cell lysate was centrifuged at high speed for 10 min at 4°C, and the supernatant was transferred to a fresh tube. The total protein content was quantified with a Bradford assay, and a total of 20 μg of protein was mixed with Laemmli buffer (4% SDS, 5% 2-mercaptoethanol (BME), 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl, pH 6.8) and denatured for 10 min at 95 ^oC. This was followed by wet transfer to PVDF immobilon membrane (Millipore IPFL00010) and blocked with Odyssey TBS blocking buffer (LiCor 927-50000). The following primary antibodies were used in the listed concentrations: LC3 (Novus Biologicals NB100-2220, 1:500) and GAPDH (Sigma–Aldrich G9545, 1:10 000). The LiCor IR secondaries were used 1:10 000 or 1:20 000 (LiCor 926-32211, 926-68071, and 926-32210). Bands were quantified and normalized by a ratio of LC3-II:GAPDH, rather than LC3-II:LC3-I as LC3-II is more sensitive in blotting and accurate observed alone.^{8 (link)}