1.4 na objective

Transfected HEK293 cells were examined with an SP8 confocal fluorescence microscope using 63 × 1.4 NA objective, Leica (Wetzlar, Germany). Alternatively, cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective and images were captured with a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by manufacturer provided software. To generate images for quantitative analysis, we kept camera, microscope, and excitation light source (X-Cite 120LEDBoost, Excelitas Technologies, 2% intensity) settings constant. To observe cells with low-level fluorescence, excitation light was set at 15% of intensity. Fluorescence intensities were obtained using Fiji^{37 (link)}. Regions of interest were used to obtain average intensity values from the transfected HEK293 cells (I_cell) and an area without cells (I_background) within the same image. Fluorescence intensity of transfected HEK293 cells (I_norm) for each cell was normalized (I_norm = I_cell − I_background). Each data sample represent average normalized intensity of the transfected cells within an image. We captured 3 to 6 fields of view per dish, using two dishes per construct in every experiment.

SK-N-BE(2) cells were obtained from Dr. B. Förthmann (MHH, Hannover, Germany). The cells were maintained in Corning plastic ware in DMEM/F12 medium supplemented with 10% FCS, glucose (4.5 g/liter), sodium pyruvate (1 mm), and penicillin-streptomycin (100 units/ml). The cells were seeded on 12-mm glass coverslips and differentiated with 10 μm all-trans-retinoic acid for a period of 6 days. The cell were fixed with 1% PFA in incomplete DMEM, permeabilized for 5 min in 0.1% Triton X-100, and blocked for 1 h with 2% BSA in PBS. Samples were incubated with rabbit polyclonal NM-2B primary antibody (Covance, catalog no. PRB-445P) for 1 h at room temperature in blocking solution, in combination with the following sheep polyclonal Tpm specific antibodies: γ/9d (Merck Millipore, catalog no. AB5447), α/1b (Merck Millipore, catalog no. ABC499), and α/9d (Merck Millipore, catalog no. AB5441) (1 (link)). Secondary antibodies were applied for 30 min at room temperature in blocking solution: GAR-IgG-AlexaFluor488 (Jackson ImmunoResearch, catalog no. 111-545-144) and DAS-IgG-Cy3 (Merck Millipore, catalog no. AP184C). Images were acquired using a Leica TCS SP8 confocal laser microscope equipped with a 63×/1.4 NA objective (Research Core Unit for Laser Microscopy, MHH). Image analysis was performed with the Fiji release of ImageJ software version 1.49s (59 (link)).

Transfected HEK293 cells were examined with an SP8 confocal fluorescence microscope using 63 × 1.4 NA objective, Leica (Wetzlar, Germany) as described previously^{17 (link)}. Briefly, cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective and images were captured using a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by Leica LAS X version 3.5 software (available at: https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). All figures were created using Adobe Illustrator CS6 (Available at: https://adobe.com/products/illustrator), under an Adobe Inc., Creative Cloud Desktop 2019 shared device license to Case Western Reserve University (CWRU) that operates until 3/31/2022.