Chapter 6 202 overnight at 40V and 4˚C in transfer buffer (15 mmol/L Tris Base, 120 mmol/L Glycine, 20% Methanol). After transfer the blot was rinsed using washing buffer (1x TBS and 0.05% Tween P1379, Sigma-Aldrich®) and imaged with the GelDoc EZ Gel Imaging System. Next, the blot was blocked using 5% non-fat milk (A0830, PanReac AppliChem) in washing buffer for one hour at RT. After blocking the primary antibodies Pol IIIA (AA: 607-698, PA558170, Invitrogen™) or Pol IIIB from Bioscience (AA:1083-1133, A301-855A) were incubated in a 1:1000 dilution in 3% Milk in washing buffer for 3 hours at RT. Next, the blots were washed with washing buffer 3 times every 10 minutes. The secondary antibody HRP (# 7074S, Cell Signaling Technology®) was then added in a dilution of 1:2000 in washing buffer and incubated for 2 hours at RT. Following the secondary antibody, the blots were washed 3 times every 10 minutes with washing buffer. The stained blots were then prepared for detection using Pierce™ ECL Western Blotting Substrate (#34095, Thermo Scientific™) following manufacturers protocol and visualized with the Odyssey® XF Imaging System (LICOR). Western blots were quantified by measuring the intensity of each band using the ImageJ software. The intensity of each band was first corrected for background intensity to remove any non-specific signal. Additionally, the band intensity was normalized to the total protein level to ensure accurate comparison between samples. A standard sample was included on every blot, serving as a control to correct for variations between different blots, ensuring consistency in quantification across all experiments. Immunocytochemistry For the localization of the target proteins subunit A and subunit B, immunocytochemistry was performed. NES cells were plated in densities of 10k cells/cm2. The cells were fixated with 2% paraformaldehyde (PFA, #15710-S, Electron Microscopy Sciences) in PBS for 20 minutes at RT. Followed by a 4% PFA fixation for another 20 minutes at RT. After fixation the cells were washed with PBS 3 times every 10 minutes. Blocking buffer consisting of 5% Normal goat serum (16210064, Gibco) 0.3% TritonX100 (T8787, Sigma-Aldrich) and 0.1% BSA (bovine serum albumin, A9418, Sigma-Aldrich) was then added to the cells and incubated for 1 hour at RT. The primary antibody Pol III A (Amino acid: 607-698, PA5-58170, Invitrogen™) or Pol III B (Amino acid: 1083-1133, A301-855A, Bioscience) was then added in a dilution of 1:500 in blocking buffer and incubated for 2 hours at RT. After incubation the cells were washed with PBS 3 times every 10 minutes. The secondary antibody was added in a dilution of 1:1000 in blocking buffer and incubated for 2 hours at RT. Following the secondary antibody, the cells were washed with PBS 3 times every 10 minutes. Lastly, DAPI
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