A1r hd confocal microscope system

For immunofluorescence analysis, self-renewing or differentiated iPSCs or hESCs were seeded on Matrigel-coated coverslips using accutase, fixed with 4% formaldehyde in PBS for 20 min, permeabilized with 0.5% Triton in PBS for 10 min, blocked in 2% BSA for 1 h and stained with indicated primary and secondary antibodies and/or Hoechst 33342 for 1 h. Images were taken using a Nikon A1R + HD confocal microscope system (Nikon Instruments, Melville, NY). 488 nm, 561 nm, and 640-nm laser lines provided illumination for hoechst, AF 488, Rhodamine Red X and AF647 fluorophores, respectively. Data were acquired using Galvano mode at 1024 × 1024 with no line averaging A Z-piezo stage (Physik Instrumente USA, Auburn, MA) allowed for rapid imaging in Z every 1 µm over an 8-µm Z distance. NIS-Elements (Nikon, Melville, NY) controlled all equipment. All images were maximum intensity projections and processed using ImageJ/FIJI.

To visualize UBA1 subcellular localization, HEK293T cells were transiently transfected with indicated pHAGE-UBA1-Flag-HA constructs. Cells were fixed with 4% formaldehyde in PBS for 20 min, permeabilized with 0.5% Triton in PBS for 10 min, blocked in 2% BSA for 1 h and stained with primary anti-UBA1a/b antibodies (Cell Signaling, 4891S, 1:500) for 1 h. After washing with PBS, cells were incubated with Alexa488-labeled donkey-anti-mouse IgGs (Jackson, Cat#:715-545-150, 1:200), rhodamine-labeled phalloidin (ThermoFisher, R415, 1:1000) and Hoechst 33342 for 1 h. Cells were mounted on slides with SlowFade Gold (Invitrogen). Images were taken using a Nikon A1R + HD confocal microscope system (Nikon Instruments, Melville NY). 488 nm, 561 nm, and 640 nm laser lines provided illumination foechsthst, AF 488, Rhodamine Red X, and AF647 fluorophores, respectively. Data were acquired using Galvano mode at 1024 × 1024 with no line averaging A Z-piezo stage (Physik Instrumente USA, Auburn MA) allowed for rapid imaging in Z every 1 µm over an 8-µm Z distance. NIS-Elements (Nikon, Melville, NY) controlled all equipment. All images were maximum intensity projections and processed using ImageJ/FIJI.

To visualize UBA1 subcellular localization, HEK293T cells were transiently transfected with indicated pHAGE-UBA1-Flag-HA constructs. Cells were fixed with 4% formaldehyde in PBS for 20 min, permeabilized with 0.5% Triton in PBS for 10 min, blocked in 2% BSA for 1h and stained with primary anti-UBA1a/b antibodies (Cell Signaling, 4891S, 1:500) for 1h. After washing with PBS, cells were incubated with Alexa488-labeled donkey-anti-mouse IgGs (Jackson, Cat#:715–545-150, 1:200), rhodamine-labeled phalloidin (ThermoFisher, R415, 1:1000) and Hoechst 33342 for 1 hour. Cells were mounted on slides with SlowFade Gold (Invitrogen). Images were taken using a Nikon A1R+ HD confocal microscope system (Nikon Instruments, Melville NY). 488 nm, 561 nm and 640 nm laser lines provided illumination f25oechsthst, AF 488, Rhodamine Red X and AF647 fluorophores, respectively. Data were acquired using Galvano mode at 1024X1024 with no line averaging A Z-piezo stage (Physik Instrumente USA, Auburn MA) allowed for rapid imaging in Z every 1 μm over an 8-μm Z distance. NIS-Elements (Nikon, Melville, NY) controlled all equipment. All images were maximum intensity projections and processed using ImageJ/FIJI.