Dylight 488 or 594 conjugated secondary antibody

Immunofluorescence staining was performed as previously described (Dridi et al., 2012 (link); Greene et al., 2020 (link)). Briefly, IPEC-J2 cells were grown in chamber slides, exposed to 37 or 54°C as described above and fixed with methanol for 10 min at −20°C before being permeabilized with Triton-X 100. Cells were blocked with serum-free protein block (Dako, Carpinteria, CA, USA) for 1 h at room temperature, then incubated with anti-GLUT1, anti-GLUT2, anti-GLUT3, anti-HSP60 (Santa Cruz Biotechnology, Dallas, TX, USA), or anti-HSP70 (Pierce Thermo Scientific, Rockford, IL, USA). All antibodies were diluted at 1:200, in antibody diluent (Dako, Carpinteria, CA, USA) and incubated overnight at 4°C. Signal was visualized with DyLight 488- or 594-conjugated secondary antibody (Thermo Fisher Scientific, Grand Island, NY, USA). Slides were coverslipped with vectashield with diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), and images were obtained and analyzed using Zeiss Imager M2 and AxioVision software (Carl Zeiss Microscopy).

Cells were fixed in 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.5% Triton X-100 for 10 min. Subsequently, cells were rinsed three times with PBS, and blocked in blocking buffer (0.1% BSA in PBS) for 30 min. Cells were incubated overnight with the specific primary antibodies at 4 °C, and washed three times with PBS. After being incubated with DyLight 488- or 594-conjugated secondary antibodies (Thermo Scientific), cells were mounted with Fluoroshield Mounting Medium with DAPI (Abcam). Fluorescent images were visualized using a Leica DMi 8 fluorescence microscope (Leica, Wetzlar, Germany). For live cell imaging, cells were grown on glass bottom dishes and treated with CLIP3 siRNA or IR. Intracellular GFP-tagged GLUT3 dynamics in live cells were imaged in an environmentally controlled chamber at 37 °C for the indicated times using LSM 800 confocal microscope (ZEISS, Oberkochen, Germany). The images were analyzed with ZEN software (ZEISS). For analysis of all microscopy images, raw image data were used.