Cells were lysed in 125 µl RIPA buffer for 20 minutes at 4°C. Proteins were extracted as previously
described (Blanco-Sánchez et al., 2014 (link)). After centrifugation at 5000 rpm for 5
minutes, the supernatant was incubated overnight at 4°C with either mouse anti-GSH (Virogen), rabbit anti-Ush1c (Novus
Biologicals) or rabbit anti-GFP (Torrey Pines Biolabs). Proteins were precipitated with protein A-Sepharose beads, washed in
lysis buffer and eluted in lysis buffer and non-reducing sample buffer (1:1). The eluted proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Detection was performed using rabbit anti-Harmonin
(Ush1c, Novus Biologicals, 1:600), mouse anti-HA (Covance, 1:500), rabbit anti-GFP (Torrey Pines Biolabs, 1:250), and mouse
anti-GSH (Virogen, 1:500) antibodies and the Odyssey Western Blotting Kit I LT (LI-COR) following the manufacturer
recommendations.
described (Blanco-Sánchez et al., 2014 (link)). After centrifugation at 5000 rpm for 5
minutes, the supernatant was incubated overnight at 4°C with either mouse anti-GSH (Virogen), rabbit anti-Ush1c (Novus
Biologicals) or rabbit anti-GFP (Torrey Pines Biolabs). Proteins were precipitated with protein A-Sepharose beads, washed in
lysis buffer and eluted in lysis buffer and non-reducing sample buffer (1:1). The eluted proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Detection was performed using rabbit anti-Harmonin
(Ush1c, Novus Biologicals, 1:600), mouse anti-HA (Covance, 1:500), rabbit anti-GFP (Torrey Pines Biolabs, 1:250), and mouse
anti-GSH (Virogen, 1:500) antibodies and the Odyssey Western Blotting Kit I LT (LI-COR) following the manufacturer
recommendations.