IRE1α mNeon-Green USOS

Description

Technical info

Tilt Series Date: 2018-10-15

Data Taken By: Stephen Carter

Species / Specimen: U2OS cells

Strain: IRE1α-mNeonGreen expression

Tilt Series Settings: Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 120.0 eV/Ų, defocus: -10.0 μm, magnification: 34000x.

Microscope: Caltech Polara

Acquisition Software: Undefined

Upload Method: reupload_downloaded

Processing Software Used: IMOD

Collaborators and Roles: Peter Walter

Purification / Growth Conditions / Treatment: Prior to cell plating, gold Quantifoil London finder grids (EMS R2/2 LF-Au-NH2) were UV treated to sterilize and coated with cell adhesion matrix. For MEFs-IRE1-mNG cell line, grids were coated in droplets of 500 ug/mL Fibronectins (Sigma-Aldrich S5171-.5MG) for 5 minutes on each side, washed in PBS, blotted and air-dried. For U2OS-IRE1-mNG cell line, grids were coated with ~4 mg/mL undiluted Collagen type I (Corning 354236) droplets for 20 minutes, washed in PBS, blotted and air-dried. Cells were seeded at 15% confluence and allowed to adhere for 8 hours and induced with Doxycycline (500 nM) for 6 or 18 hours for MEFs and U-2 OS cell lines, respectively. ER stress was then induced by treatment with 1.5 ug/mL of Tunicamycin for 2 hours.

Sample Preparation: Manual Blotting

Attached Files

tomography5.mrc, Tilt Series (Pixel Size 6.52 nm), 859.7 MB

tomography5_orig_full.rec, Reconstruction (Pixel Size 0.0 nm), 8.9 GB

sirt.rec, Reconstruction (Pixel Size 0.0 nm), 9.2 GB