Transcription factor antagonism regulates heterogeneity in embryonic stem cell states
This folder contains supplemental files for "Transcription factor antagonism regulates heterogeneity in embryonic stem cell states". The available files are: (1) immunofluorescence images of Klf4 and Zfp281 in mESC (2) images Klf4 and Zfp281 immunoblots in mESC
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Immunofluorescence: V6.5 ESC were stained for Klf4 and Zfp281 in the following manner. Anti-Klf4 was conjugated with Alexa Flour 488 using Alexa Fluor Antibody Labeling Kit and following manufacturer’s instructions (Thermo Fisher A20181). 50 uL of 1mg/mL anti-Klf4 (abcam ab129473) was used for conjugation. Unconjugated anti-Zfp281 (abcam ab101318) was used. Cells were plated onto gelatinized, glass coverslips at least 1 day prior to fixation. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, washed three times with PBS, and permeabilized and blocked with 0.3% Triton X-100 in 0.1M glycine and 1% rabbit serum (Jackson ImmunoResearch 011-000-120) for 30 minutes, and washed three times with PBS. Cells were stained overnight at 4C with anti-Zfp281 primary antibody (1:1000) in 1% BSA and 1% rabbit serum in PBST (0.1% Tween 20 in 1xPBS). The next day, cells were washed three times with PBS and incubated with goat anti-rabbit Alexa Flour 657 (1:1000, Thermo A-21245) in 1% BSA in PBST for 2 hours in the dark at room temperature. Cells were washed three times with PBS, and then incubated with 10% rabbit serum in 1% BSA in PBST for 1 hour in the dark at room temperature to saturate anti-rabbit binding sites. Next, cells were stained with Klf4*488 (1:250) in 1% BSA and 1% rabbit serum in PBST overnight at 4C. The next day, cells were washed three times with PBS and incubated with 0.5ug/mL DAPI for 5 minutes in the dark at room temperature. Coverslips were washed three times with PBS and mounted with Vectashield with DAPI (Vector H-1200-10). Mounted coverslips were examined with Nikon A1R Spectral Scanning Confocal microscope. All images were taken with the same microscope settings and processed with ImageJ. Alexa Fluor 488 was imaged at an excitation of 465-495 nm and emission of 515-555 nm. Alexa Fluor 657 was imaged at an excitation of 625-650 nm and emission peak of 670 nm. Immunoblot: WT, Klf4-/-, and Zfp281-/- mESC were washed twice with cold PBS, harvested via scraping, and pelleted at 4C. Cells were resuspended in 200 μL RIPA lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] supplemented with protease inhibitor cocktail (cOmplete, Roche) and lysed under constant agitation at 4oC for 30 minutes. Lysates were pelleted at 4C, and 30 μg of protein was mixed with 2x Laemmli buffer with B-Mercaptoethanol and boiled for 5 min at 100 temperature. Samples were loaded and run on a 12% SDS-polyacrylamide electrophoresis gel, transferred onto nitrocellulose membrane, and immunoblotted using anti-Klf4 (1:1000, R&D AF3158) or anti-Zfp281 (1:500, Abcam 101318) and anti-GAPDH (1:1000, Ambion AM4300) antibodies. Membranes were probed using anti-goat 800nm (1:10,000, LI-COR 926-32214) or anti-rabbit 800nm (1:10,000, LI-COR 925-32213) and anti-mouse 700nm (1:10,000, LI-COR 925-68072) and imagined on LI-COR Odyssey.