Alexa488 or alexa594 conjugated anti mouse or anti rabbit antibodies

HeLa cells or NIH-3T3 cells were cultured on μ-dish (ibidi, GmbH, Am Klopferspitz, Germany) and transiently transfected either with shRNA SigR1 alone or together with GFP-LC3 constructs. The medium was changed after 4 h. After 48 h, cells were fixed in 4% PFA and processed for confocal microscopy. In further experiments after 48 h of knockdown, cells were permeabilized with 0.1% Triton X-100, fixed in 4% PFA and immunostained (see Supplementary Table 1 for antibody dilutions). Secondary Alexa488- or Alexa594-conjugated anti-mouse or anti-rabbit antibodies (Invitrogen) were used for visualization. Nuclei were stained with Hoechst 33342 (1 μg/ml). Samples were mounted with fluorescent mounting media (DAKO, Glostrup, Denmark) and visualized using a Zeiss LSM 700 confocal microscope (Zeiss, Göttingen, Germany). Resulting images were processed using the Zeiss LSM software and Adobe Photoshop CS5 (Adobe Systems GmbH, Munich, Germany).

HeLa, MCF-7, Cos-7, MEF and NIH-3T3 cells were cultured on μ-dishes (ibidi, GmbH, Planegg/Martinsried, Germany) and transiently transfected either with wtSigR1 or mSigR1. After 48 h cells were fixed in 4% PFA and processed for confocal microscopy. Permeabilization with 0.5% Triton X100 and blocking with 4% skimmed milk/goat serum was followed by primary antibody incubation overnight at 4 °C. Secondary Alexa488- or Alexa594-conjugated anti-mouse or anti-rabbit antibodies (Invitrogen) were used for visualization. Nuclei were stained with Hoechst 33342 (1 μg/ml) or were mounted with DAPI containing fluorescent mounting media (DAKO) and visualized using a Zeiss LSM 700 confocal microscope. Resulting images were processed using the Zeiss LSM software and Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA).