Aclar sheets

Lc3b-AP2-expressing imMEFs were grown on aclar sheets (Science Services), supplemented with 4.83 µM Hemin chloride (ROTH) for 16 h and treated with 200 nM BafA1 (Biomol) for 2 h before fixation. Cells were fixed in 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M sodium cacodylate buffer (pH 7.4; CB) for 30 min. Fixation and the following processing steps were carried out on ice. After washes in CB, endogenous peroxidases were blocked in 20 mM glycine (Sigma) in CB for 5 min and cells washed in CB. 1x diaminobenzidine (DAB) in CB with 2 mM calcium chloride was prepared from a 10x DAB stock (Sigma) in hydrochloric acid (Sigma) and added to the cells for 5 min without and for another 40 min with 10 mM H₂O₂ (Sigma). After washes in CB, cells were postfixed in reduced osmium (1.15% osmium tetroxide, Science Services; 1.5% potassium ferricyanide, Sigma) for 30 min, washed in CB and water and incubated over-night in 0.5% aqueous uranylacetate (ScienceServices). Dehydration was accomplished using a graded series of ice-cold ethanol. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. Cells were ultrathin sectioned at 50 nm on formvar-coated copper grids (Plano). TEM images were acquired on a JEM 1400plus (JEOL) using the TEMCenter and Shotmeister software packages (JEOL) and analysed in Fiji.

293T cells expressing APEX2-tagged TECPR2 WT and L440Rfs were grown on aclar sheets (Science Services) and fixed with 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M pH 7.4 sodium cacodylate buffer (CB) for 30 min on ice. Endogenous peroxidases were blocked with 20 mM glycine (Sigma) for 5 min on ice and cells washed in CB. Cells were saturated with freshly prepared 1× diaminobenzidine (DAB, in CB supplemented with 2 mM calcium chloride) for 5 min and APEX2 activity was triggered with 1× DAB supplemented with 10 mM H₂O₂ (Sigma) for 40 min on ice. Cells were washed with CB and subsequently postfixed and contrasted in reduced osmium (1.15% osmium tetroxide (Science Services) 1.5% potassium ferricyanide (Sigma)) for 30 min. After washes in CB and H₂O₂, cells were incubated in 0.5% aqueous uranylacetate (Science Services) over-night and dehydrated using a graded series of ice-cold ethanol-water composite. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. 50 nm ultrathin sections were generated on formvar-coated copper grids (Plano). Sections were imaged using a JEM-1400 + (JEOL) equipped with a XF416 (TVIPS) and the EM-Menu software (TVIPS) and analyzed using ShotMeister (JEOL).